While the signaling pathways and transcription factors active in adult slow- and fast-type muscles begin to be characterized, genesis of muscle fiber-type diversity during mammalian development remains unexplained. We provide evidence showing that Six homeoproteins are required to activate the fast-type muscle program in the mouse primary myotome. Affymetrix transcriptomal analysis of Six1(-/-)Six4(-/-) E10.5 somites revealed the specific down-regulation of many genes of the fast-type muscle program. This data was confirmed by in situ hybridization performed on Six1(-/-)Six4(-/-) embryos. The first mouse myocytes express both fast-type and slow-type muscle genes. In these fibers, Six1 and Six4 expression is required to specifically activate fast-type muscle genes. Chromatin immunoprecipitation experiments confirm the binding of Six1 and Six4 on the regulatory regions of these muscle genes, and transfection experiments show the ability of these homeoproteins to activate specifically identified fast-type muscle genes. This in vivo wide transcriptomal analysis of the function of the master myogenic determinants, Six, identifies them as novel markers for the differential activation of a specific muscle program during mammalian somitic myogenesis.