Ahmir Khalib Thompson was born into a musical family in Philadelphia on January 20, 1971. His father was Arthur Lee Andrews Thompson, from Goldsboro, North Carolina, and went North in the Great Migration. A singer, he became known as Lee Andrews and was lead with Lee Andrews & the Hearts, a 50s doo-wop group. Ahmir's grandfather Beachy Thompson had sung with the gospel group, The Dixie Hummingbirds. Ahmir's mother, Jacquelin Thompson, together with his father, was also part of the Philadelphia-based soul group 'Congress Alley'. His parents did not want to leave the boy with babysitters, so they took him on tour with them. He grew up in backstages of doo-wop shows. By the age of seven, Thompson began drumming on stage at shows, and by 13, had become a musical Director.

The Roots' lineup was soon completed, with Questlove on drums and percussion, Tariq Trotter and Malik B on vocals, Josh Abrams (Rubber Band) on bass (who was replaced by Leonard Hubbard in 1994), and Scott Storch on keyboards. While the group was performing a show in Germany, they recorded an album entitled Organix, released by Relativity Records in 1993.

The group continued recording, releasing two critically acclaimed records in 1995 and 1996, Do You Want More?!!!??! and Illadelph Halflife, respectively.

In 1999, The Roots had mainstream success with "You Got Me" (featuring Erykah Badu); the song earned the band the Grammy Award for Best Rap Performance by a Duo or Group for 2000. The song helped fuel the success of their Things Fall Apart album, which has since been hailed as a classic, eventually selling platinum.

In 2000 Questlove appeared with The Roots in Bamboozled, directed by Spike Lee.

In 2001 Questlove appeared with many other Roots members as The Lions in Marc Levin's 2001 Brooklyn Babylon, in which Black Thought had a starring role.

In 2003, he played drums on John Mayer's song "Clarity" from his second album Heavier Things. He also arranged and drummed on Joss Stone's cover of the White Stripes' "Fell in Love with a Girl".

In 2004, The Roots released The Tipping Point, which contained a more mainstream sound, allegedly due to demands from Interscope Records. The album sold 400,000 copies. In 2004, Questlove appeared in Jay-Z's Fade to Black. In addition to appearing in the documentary portion of the film, Questlove was the drummer/musical Director for all portions of the show with a live band. In 2005, Questlove appeared along with such luminaries as Madonna, Iggy Pop, Bootsy Collins, and Little Richard in a television commercial for the Motorola ROKR phone. Questlove also appears for a short clip in the 2005 film, The Longest Yard.

In 2005, Questlove appeared with The Roots in Dave Chappelle's Block Party and the following year, he appeared in the popular music film, Before the Music Dies. His latest appearances include Beats, Rhymes & Life: The Travels of a Tribe Called Quest and documentary The Black Power Mixtape 1967-1975 (2010) for which he won the Swedish Guldbagge Award for best music together with Om'Mas Keith.

In 2006, Questlove appeared in the film Dave Chappelle's Block Party, as well as a couple of skits on Chappelle's Show. These included the Tupac "The Lost Episodes" skit, and one featuring John Mayer, wherein Questlove performs in a barber shop, inducing the occupants to dance and rap. With the exception of The Fugees and Jill Scott, Questlove served as the Drummer at the 2004 Brooklyn street concert and was the musical Director for the entire show. Questlove was given an Esky for Best Scribe in Esquire magazine's 2006 Esky Music Awards in the April issue. In 2006, Questlove was one of a handful of Musicians hand-picked by Steve Van Zandt to back Hank Williams Jr. on a new version of "All My Rowdy Friends Are Coming Over Tonight" for the season premiere (and formal ESPN debut) of Monday Night Football. Along with his fellow Motorola ROKR commercial co-stars, Bootsy Collins and Little Richard, Questlove's bandmates included Rick Nielsen (Cheap Trick), Joe Perry (Aerosmith), Charlie Daniels, and Bernie Worrell. In the same year he appeared in the studio album Fly of the Italian singer Zucchero Fornaciari.

In 2007, Questlove co-produced with VH1's The Score winning Producer Antonio "DJ Satisfaction" Gonzalez, from The Maniac Agenda, the theme to VH1's Hip Hop Honors 2007. Questlove joined Ben Harper and John Paul Jones for the Bonnaroo SuperJam on June 16, 2007, to play a 97-minute set. In 2007 he provided the foreword for the book Check the Technique.

In late 2009, while serving as an associate Producer of the hit Broadway play Fela!, Questlove recruited Jay-Z to come on board as a Producer. It was reported that Will Smith and Jada Pinkett Smith have also signed on as producers.

In January 2010, he was writing material with British singer Duffy for her second album. He has been featured in a commercial for Microsoft's short-lived mobile phone, the Kin. In 2010, he made a cameo in the music video of Duck Sauce's song "Barbra Streisand", and with The Roots released the album Dilla Joints with renditions of Producer J Dilla's music. He contributed drums to the song "You've Got a Lot to Learn", which was recorded for the self-titled third studio album by Evanescence, but did not appear on the final release.

Questlove was planning to collaborate with Amy Winehouse before her death in July 2011. He said "We're Skype buddies, and she wants to do a project with Mos and me. Soon as she gets her visa thing together, that's gonna happen." Rolling Stone named Questlove #2 in the 50 Top Tweeters in Music. In June 2011, Questlove played drums alongside The Roots Bassist Owen Biddle for Karmin's cover of Nicki Minaj's "Super Bass." Questlove placed 8th in the Rolling Stone Readers Pick for Best Drummers of all Time.

In 2013, Questlove appeared as himself on The Eric Andre Show as a part of the show's band.

In 2014, Questlove appeared in the famous James Brown documentary Mr. Dynamite: The Rise Of James Brown that was directed by Alex Gibney

In 2015, Questlove appeared on Parks and Recreation as Levondrious, the estranged brother of Retta's character Donna Meagle. Questlove also appeared in a documentary film that depicts the life and death of Amy Winehouse called Amy.

In 2016, Questlove appeared in the film Popstar: Never Stop Never Stopping as himself.

In December 2017, Questlove was one of the subjects of an episode the TV series Finding Your Roots, produced by Henry Louis Gates. He learned that one set of 3xgreat-grandparents had been taken captive in Ghana, sold as slaves to traders in Dahomey, and brought to the US illegally in 1860 on Clotilda, which had been adapted as a slave ship.