NBC moved Jay Leno's retirement and Fallon's takeover date up from September 2014 to February 17, 2014, at Leno's suggestion, so it could use the 2014 Winter Olympics as a springboard for Fallon's launch.

The transition was reportedly accelerated because NBC feared that Jimmy Kimmel, who had recently moved to the 11:35 slot on ABC, could lock up the young demographic if the network waited too long to promote Fallon.

After his acclaimed Donald Trump impression on The Tonight Show, Fallon was asked to play Trump on the December 19, 2015 episode of Saturday Night Live because Taran Killam was busy playing Ted Cruz, but the plan fell through and Darrell Hammond played the role instead.

NBC spent approximately $5 million renovating Studio 6B, the same studio used for The Tonight Show Starring Johnny Carson, increasing its seating capacity from 189 to around 240 and adding a new control room and lobby.

Because Studio 6B and Seth Meyers' Studio 8G sit directly on top of each other in a building with steel girders that conduct sound between floors, The Tonight Show tapes at 5:00 p.m. and Late Night tapes later at 6:30 p.m. to avoid sound bleed.

On March 22, 2021, Fallon became the first major American late-night host to resume taping in front of a studio audience during the COVID-19 pandemic, with the first week's audience limited to just 58 frontline workers.

On April 1, 2022, Jimmy Fallon and Jimmy Kimmel swapped shows as an April Fools' Day prank, with Fallon hosting Jimmy Kimmel Live! from Los Angeles and Kimmel hosting The Tonight Show from Studio 6B, with only a small number of producers, writers and executives informed beforehand.

The show's opening sequence was directed by filmmaker Spike Lee and features Fallon visiting New York City landmarks like Grand Central Terminal and Katz's Delicatessen, while the show's logo featuring a full moon is a callback to The Honeymooners.

A 2023 Rolling Stone exposé featured testimony from sixteen former and current staff members alleging Fallon was drunk on set and abrasive toward writers, with staffers using dressing rooms as 'crying rooms' and one admitting to suicidal thoughts due to his behavior.

The show's popular 'Lip Sync Battle' segment was spun off into its own series after NBC passed on it, not realizing its potential despite Emma Stone's viral rendition of DJ Khaled's 'All I Do Is Win.'