Pinky and the Brain was created by Tom Ruegger as a collaboration between Warner Bros. Television Animation and Steven Spielberg's Amblin Entertainment, first appearing as a recurring segment on Animaniacs in 1993 before being spun off into its own series due to popularity.

The characters were inspired by two real producers of Tiny Toon Adventures, Eddie Fitzgerald and Tom Minton, who shared an office and developed quirky personalities: Minton seemed always scheming to take over the world while Fitzgerald injected nonsense words like 'Narf' and 'Egad'.

During Animaniacs' development, animator Bruce Timm drew caricatures of Fitzgerald and Minton, and Tom Ruegger added mouse ears and noses to the drawings, cementing the visual concept for Pinky and the Brain.

Maurice LaMarche, a big fan of Orson Welles who had already voiced Welles in the 1994 film Ed Wood, based the Brain's voice on Welles during his audition and was cast on the spot; he describes the voice as '65% Orson Welles, 35% Vincent Price'.

Rob Paulsen, already voicing Yakko Warner on Animaniacs, was chosen to voice Pinky and drew inspiration from British comedies like Monty Python's Flying Circus, The Goon Show, and Peter Sellers to create Pinky's exaggerated British accent.

An episode parodying Orson Welles' infamous 'Frozen Peas' outtake was secretly written by the show's writers using LaMarche's actual sound-check lines; the recording was deliberately scheduled on the same day as the funeral of Sam Kinison, LaMarche's close friend, and he broke down in happy tears upon recognizing the in-joke script, which writer Peter Hastings called 'a $250,000 inside joke'.

Brain actually meets a caricature of Orson Welles working as a Hollywood busboy in the episode 'What Ever Happened to Baby Brain,' and the two end up shouting in unison, 'Things will be different when I take over the world!'

The character Larry was added only in response to pressure from Kids' WB executives who wanted more characters, but the writers deliberately kept his appearances sporadic since they felt a third character would ruin the Pinky and Brain comedy duo dynamic; his debut episode even mockingly reworked the theme song to include 'and Larry!'

The 1998 retooling into Pinky, Elmyra & the Brain, forced by network pressure and placing the mice under Tiny Toons character Elmyra Duff, was poorly received by the crew; Rob Paulsen noted that Spielberg had wanted the Tiny Toons and Animaniacs universes kept separate, and the new format reduced Pinky to the useless 'Larry' role of that series.

The episode 'Inherit the Wheeze,' about Brain suffering the effects of smoking due to a tobacco company, won a PRISM Award for its anti-smoking message.

Naughty Dog co-founder Jason Rubin was a fan of the show, and the Brain served as an influence in the creation of Doctor Neo Cortex, the main antagonist of the Crash Bandicoot video game series.

The show's score sometimes references classical music, such as Dvořák's New World Symphony playing during an episode where Brain builds a papier-mâché Earth, and Mussorgsky's Pictures at an Exhibition in the episode where Pinky becomes the artist 'Pinkasso'.