The complex chemical environment required for the development of the myofiber (myotube) from embryonic avian muscle myoblasts in vitro has been simplified. Myotube formation is shown to occur in the presence of insulin, a low-molecular-weight (fusion) factor obtained from embryo extract, and a collagen substratum. In the absence of collagen, globular structures are formed which are microscopically identical to the globular syncytial structures previously described [de la Haba and Amundsen (1972) Proc. Nat. Acad. Sci. USA 69, 1131-1135]. A high-molecular-weight fraction from embryo extract will also promote myotube formation in the presence of the fusion factor. This fraction is shown to contain (a) collagen, which promotes myotube formation, and (b) an additional factor which also promotes myotube formation, and which we tentatively hypothsize to be an inducer of collagen synthesis by myogenic cells.