After completing high school, Poston attended Bethany College in West Virginia, but did not graduate. While there, he joined the Sigma Nu Fraternity. He joined the United States Army Air Forces in 1941. Accepted to officer candidate school and then graduating from FLIGHT training, Poston served as a pilot in the European Theater in World War II; his aircraft dropped paratroopers for the Normandy invasion.

In 1953, as Thomas Poston, he was cast as "Detective" in the film City That Never Sleeps. In 1957, Poston gained recognition as a comedic "Man in the Street" (along with his colleagues Louie Nye, Dayton Allen and Don Knotts) on The Steve Allen Show. For these performances, Poston won the 1959 Emmy Award for Best Supporting Actor (Continuing Character) in a Comedy Series. In the fall of 1959, when the Allen program moved west to Los Angeles, Tom remained in New York, appearing frequently on Broadway and television game shows.

Poston and Actress Jean Sullivan were married in 1955 and divorced in 1968. Their daughter is Actress Francesca Poston.

However, his television career was expansive, covering the better part of five decades. When Mel Brooks submitted his idea for Get Smart to the ABC network, ABC wanted Poston for the lead role of Maxwell Smart. When ABC passed on the show, NBC picked it up and the lead went to Don Adams. Poston, however, made a guest appearance on the show as a KAOS villain. He appeared in Thriller during its second season in 1961. The episode, number six, was entitled "Masquerade" and starred Elizabeth Montgomery.

His film career was limited, with appearances in films such as william Castle's Zotz! (1962), The Old Dark House (1963), Soldier in the Rain (1963), Cold Turkey (1971), The Happy Hooker (1975), Rabbit Test (1978), Up the Academy (1980) and Carbon Copy (1981).

Poston married Kay Hudson in 1968. They had two children, daughter Hudson Poston and son Jason Poston. They divorced in 1975 but remarried in 1980 and remained together until her death in 1998 from ALS.

Poston was a recurring guest star on The Bob Newhart Show in the 1970s. He later played the role of Franklin Delano Bickley on Mork & Mindy.

According to USA Today Life Editor Dennis Moore, Poston appeared in more sitcoms than any other actor. In the 1980s, he played George Utley opposite Bob Newhart's character on Newhart.

A longtime friend of Bob Newhart, Poston played George Utley, a simple country handyman of the Stratford Inn, on Newhart and appeared with Newhart in Cold Turkey as the town drunk, Edgar Stopworth. He was nominated for an Emmy Award three times for Outstanding Supporting Actor in a Comedy Series for his performance on Newhart in 1984, 1986, and 1987. He had a third role with Newhart in the short-lived Bob.

Poston had regular roles on many other television series: Family Matters, Murphy Brown, Home Improvement, Cosby, Malcolm & Eddie, ER, Grace Under Fire, That '70s Show (as Kitty Forman's father, Burt Sigurdson), Will & Grace, and guest starred in an episode of The Simpsons as the Capital City Goofball. He played dentist/jeweler, Art Hibke, on ABC's Coach, for which he was nominated for an Emmy for Outstanding Guest Actor in a Comedy Series in 1991. He guest-starred on Home Improvement as a surly airport clerk in Alpena, Michigan when Tim and Al get stuck there during a snowstorm on Christmas Eve, and again as that character's brother in the episode "The Tool Man Delivers", and again as the third brother in the episode “Thanksgiving”.

In 2001, Poston married Actress Suzanne Pleshette, who played the wife of Newhart's character Bob Hartley on The Bob Newhart Show, his fourth marriage, and her third marriage.

After a brief illness, Poston died of respiratory failure on April 30, 2007, in Los Angeles, California at the age of 85. He predeceased his widow, Susan Pleshette, by 7 months. Though he was not Jewish, he is interred in the Jewish Hillside Memorial Park Cemetery alongside Pleshette, who was Jewish.