Thompson was born in Philadelphia on January 20, 1971. His father was Lee Andrews of Lee Andrews & the Hearts, a 50s doo-wop group. His mother, Jacqui Andrews, together with his father was also part of the Philadelphia based soul group Congress Alley. His parents did not want to leave him 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.

Questlove's parents then enrolled him at the Philadelphia High School for the Creative and Performing Arts. By the time he graduated, he had founded a band called The Square Roots (later dropping the word "square") with his friend Tariq Trotter (Black Thought). Questlove's classmates at the Philadelphia High School for the Creative and Performing Arts included Boyz II Men, jazz bassist Christian McBride, jazz guitarist Kurt Rosenwinkel, jazz organist Joey DeFrancesco, and singer Amel Larrieux. He attended senior prom with Larrieux.

Questlove began performing on South Street in Philadelphia using drums, while Tariq rhymed over his beats and rhythms.

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 was a cameo in the music video of Duck Sauce's song "Barbra Streisand". In 2010, In an act of great musicianship the legendary Roots-crew pay respect to the late and great J Dilla offering their renditions of the producer’s greatest hits. The featured mixtape belongs easily to the most sublime tributes to J Dilla we have seen to date. Enjoy in this highest form of appreciation, the album is called Dilla Joints. He contributed drums to the song "You've Got a Lot to Learn", a single on Evanescence's self-titled third album. 2011 - Guest DJ at LA Food & Wine's Decadence after party

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 YouTube sensation Karmin's cover of Nicki Minaj's Super Bass. Questlove placed 8th in the Rolling Stone Readers Pick for Best Drummers of all Time.

He currently performs with The Roots on Late Night with Jimmy Fallon, occasionally performing solos titled 're-mixing the clips' where he draws on his production and DJ abilities to dub video clips, cue audio samples in rhythm, and play drum breaks simultaneously.