Generation of multiple cell types from embryonic stem (ES) cells and induced pluripotent stem cells is crucial to provide materials for regenerative medicine. EGAM1N has been found in preimplantation mouse embryos and mouse ES cells as a functionally unclassified homeoprotein. Recently, we reported that expression of EGAM1N suppressed the in vitro differentiation of ES cells into progenitor cells that arise in early embryogenesis. To clarify the effect of EGAM1N on terminal differentiation, embryoid bodies (EBs) were prepared from ES cells expressing exogenous Egam1n. In EBs expressing Egam1n, cardiomyogenesis was inhibited by impairing the expression of crucial transcription factors Brachyury T and Nkx2.5 in the generation of mesoderm and cardiomyocytes, respectively. Expression levels of Mef2c, another crucial gene for cardiomyogenesis, were unaffected. Conversely, the expression levels of Gata6 and Plat, markers for the primitive endoderm lineage, and Cdx2, a marker for the trophectoderm lineage, were increased. These results suggested that certain cell populations in EBs expressing Egam1n preferentially differentiated to such cell lineages. Our results suggest that EGAM1N not only affects the generation of progenitor cells during early embryogenesis, but also the progression of terminal differentiation, such as cardiomyogenesis, in mouse ES cells.