He first reached a mainstream audience through his performance on "The Ballad of Jed Clampett", the theme for the network television series The Beverly Hillbillies, in the early 1960s.

In 1943, he played mandolin and sang tenor in The Kentucky Pardners, the band of Bill Monroe's older brother Charlie.

[4] When they parted ways in 1969, Flatt formed a new group, the Nashville Grass, hiring many of the Foggy Mountain Boys.

[5] His role as rhythm guitarist and vocalist in each of these seminal ensembles helped define the sound of traditional bluegrass music.

Lester's hometown of Sparta, Tennessee, held a bluegrass festival in his honor for a number of years, before being discontinued a few years prior to the death of the traditional host, resident Everette Paul England; Lester Flatt Memorial Bluegrass Day remains part of the annual Liberty Square Celebration held in Sparta.