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A studio plot synopsis indicates that the film originally contained the following opening scene: Burlesque dancers "Dixie Daisy" and "Gee Gee Graham" perform in Columbus, Ohio. While eating breakfast in a cafeteria one morning, they read the tea leaves in the bottom of their cups, and see a trip and violent death predicted for their future. Just then, Dixie receives a telegram from burlesque impresario "S. B. Foss" inviting her to perform at his theater in New York. Dixie makes her debut a week later. As this scene was not included in the viewed print, and is not referred to in reviews, it May have been cut prior to the film's press preview. The working title of this film was G-String Murders. Information in the file on the film in the MPAA/PCA Collection at the AMPAS Library indicates that the PCA had concerns about many elements of the film. The PCA recommended the following changes: "Specifically, we are concerned about the prominent use of the object known as the 'G-String' as a murder weapon. It is our impression that the use of this extremely intimate female garment will be considered offensive...We believe the [burlesque skit] about the 'Pickle Persuader' is definitely offensive, and suggest that you consider replacing it with something else." The "Pickle Persuader" skit remains in the film. The PCA also influenced producer Hunt Stromberg to change the title from G-String Murders after a state censor board lodged a written protest.
According to a October 15, 1941 news item in Hollywood Reporter, producer David O. Selznick had taken out an option on Gypsy Rose Lee's novel for $1,000 against a $25,000 purchase price and that he was to test Lee for the starring role in the film. Other Hollywood Reporter news items indicate that Selznick was planning to loan producer John Houseman to United Artists for the production, that the book option was later picked up by United Artists for the $25,000 asking price, and that Joseph Cotten was considered for a lead role in the film. Lady of Burlesque marked Michael O'Shea's feature film debut and Hunt Stromberg's first independent production. Stromberg was previously a prominent producer at M-G-M. Famous burlesque dancer Lee, who was born Rose Louise Hovick, made her feature film debut in Twentieth Century-Fox's 1937 film You Can't Have Everything, using the name Louise Hovick (see AFI Catalog of Feature Films, 1931-40; F3.5237). Lady of Burlesque was the first film on which she was credited onscreen by her more familiar stage name, Gypsy Rose Lee. Arthur Lange was nominated for an Academy Award for Best Music (Scoring of a Dramatic or Comedy Picture) for this film.