The mouse blastocyst-derived embryonic stem cell (ES cell) line BLC6 efficiently differentiates into myosin heavy chain-, desmin- and myogenin-positive skeletal muscle cells when cultivated in embryo-like aggregates (embryoid bodies). Here, we show that the muscle-specific determination genes myf5, myogenin, myoD, and myf6 are expressed in these embryoid bodies in a characteristic temporal pattern which precisely reflects the sequence observed during mouse development in vivo. Myf5 is the first gene to be expressed followed by myogenin, myoD, and myf6, in this order. In situ hybridization demonstrates transcripts for myogenin and myoD accumulating in mono- and multinucleated myogenic cells, while myf5 mRNA is already found in mononucleated myoblasts. The myocytes also express functional nicotinic cholinoceptors and exhibit T-type Ca2+ currents and later L-type Ca2+ currents, demonstrating physiological properties of skeletal muscle cells. During myocyte differentiation the density of L-type Ca2+ channels significantly increases while the density of T-type Ca2+ channels decreases. The effect of external signals on myogenic differentiation of BLC6 cells was demonstrated by cocultivation with visceral endodermal END-2 cells and the activin A-secreting WEHI-3 cells. END-2 cells essentially prevent skeletal muscle differentiation, whereas basic fibroblast growth factor, transforming growth factor-beta, and WEHI-3 cells have no or an attenuating effect, respectively. Our results suggest that ES cells recapitulate closely the early steps of muscle development in vivo and may serve as an excellent in vitro system to study this process.