Targeted inactivation of the myogenic determination genes myf-5 and myoD in mice resulted in moderate (Myf-5) or no muscle phenotypes (MyoD) and double knock-out mutants lacking both genes failed to develop any skeletal muscle. In order to determine the mechanism of this apparent genetic redundancy we investigated the basis of functional overlap between the two genes. Here we demonstrate that Myf-5 and MyoD are not expressed within the same muscle precursor cell, but rather determine different muscle cell lineages arising from independently committed stem cell populations. Selective ablation of Myf-5-expressing muscle precursors from differentiating ES cells does not prevent Myo-D-dependent muscle differentiation. The early muscle progenitor cells which normally express Myf-5 do not develop into later appearing MyoD cells, even when the myf-5 gene has been inactivated. Thus skeletal musculature in vertebrates develops from two separate cell lineages and complementation may occur at the cellular level, but not between different myogenic factor genes within one cell.