The mammary gland is one of few adult tissues that strongly induce de novo fatty acid synthesis upon physiological stimulation, suggesting that fatty acid is important for milk production during lactation. The committed enzyme to perform this function is fatty acid synthase (FASN). To determine whether de novo fatty acid synthesis is obligatory or dietary fat is sufficient for mammary gland development and function during lactation, Fasn was specifically knocked out in mouse mammary epithelial cells. We found that deletion of Fasn hindered the development and induced premature involution of the lactating mammary gland, significantly decreased short- and medium-chain fatty acids and total fatty acid contents in the milk. Consequently, pups nursing from Fasn knockout mothers experienced growth retardation and pre-weanling death, which was rescued by cross-fostering pups to a lactating wild type mother. These results demonstrate that FASN is essential for the development, functional competence, and maintenance of the lactating mammary gland.