Epiblast cells form skeletal muscle and neurons in culture and some express mRNA for the skeletal muscle specific transcription factor MyoD in vivo. The following experiments were designed to determine whether the neurogenic transcription factor NeuroM is expressed in the epiblast and if NeuroM and MyoD are present in separate subpopulations of epiblast cells that can differentiate into neurons and muscle, respectively. In situ hybridization revealed that NeuroM was present in the anterior region of the pregastrulating epiblast. Some cells with NeuroM were proliferating and expressed two molecules present in neurogenic cells, NCAM and the Zn-12/HNK-1 carbohydrate. The G8 antibody labeled cells with MyoD but not NeuroM. When G8 positive cells were isolated by magnetic cell sorting and placed in culture, nearly all differentiated into skeletal muscle in serum free medium. A subpopulation of cells isolated with antibodies that bound to cells expressing NeuroM formed neurons when cultured in medium supplemented with sera and embryo extract. These experiments demonstrate that NeuroM and MyoD are present in separate subpopulations of cells in the pregastrulating epiblast. Epiblast cells with NeuroM are more dependent on exogenous factors to differentiate than those with MyoD.