Previously it was observed that mixture cultures of “nullipotent” and pluripotent embryonal carcinoma (EC) cells failed to differentiate, suggesting that nullipotent cells might restrict the differentiation of pluripotent cells ( M. J. Rosenstraus and A. J. Levine, 1979, Cell 17, 337–346). This report shows, however, that the differentiation of mixed cultures is dependent on the relative growth rates of the two cell types and that nullipotent cells do not directly affect pluripotent cell development. When the growth rate of nullipotent cells was modulated by taking advantage of drug-resistance markers, mixed cultures, containing pluripotent PSA-1 cells and a genetically marked subclone of nullipotent F9 cells, exhibited extensive differentiation. The differentiated cells were PSA-1 derivatives, whereas, the F9 cells remained as undifferentiated EC cells. Similar results were obtained when a genetically marked PSA-1 subclone was cocultured with a second nullipotent cell line, Nulli SSC1. Thus nullipotent and pluripotent EC cells appear to express their developmental potential autonomously in mixed cultures. This implies that the nullipotent cell lines studied are intrinsically incapable of responding to the conditions that trigger in vitro differentiation of EC cells upon aggregation.