Kightlinger began her career as a stand-up comic and has two HBO specials and six Comedy Central specials to her credit. She was a Writer/Producer on Will & Grace where she also had a recurring role as Nurse Sheila. Recently, Kightlinger was a series regular in the HBO comedy Lucky Louie, starring Louis C.K. Her book, Quick Shots of False Hope, described in the New York Times Book Review as "memorable, disturbing and darkly comic," is being adapted for film. She recently completed a mockumentary that she directed and co-wrote with Jeremy Kramer about the making of a horror film, entitled Near Lies and Half Truths: Cat Demon Re-Exhumed.

Laura's documentary, Sixty Spins Around the Sun, about a former stand-up comic struggling with addiction while fighting to repeal the Rockefeller Drug Laws, won Best Documentary at the Empire State, Beverly Hills, and Boston film festivals. It was also featured at the HBO Comedy Arts Festival and has been acquired by Netflix. Additionally, Kightlinger received the Best New Director Award from the I.F.C.T., the Black Maria Director's Citation Award for her short, Dependable People and was nominated for a Best Actress Satellite Award for her role as Jackie Woodman.