Embryonic stem (ES) cells have been considered as a valuable renewable source of materials in regenerative medicine. Recently, we identified the homeoprotein EGAM1 both in preimplantation mouse embryos and mouse ES cells. Expression of the Egam1 transcript and its encoded protein was detectable in differentiating mouse ES cells, while it was almost undetectable in undifferentiated cells. In the present study, in order to clarify the effect of forced expression of EGAM1 on the differentiation of mouse ES cells in vitro, transfectants expressing exogenous EGAM1 were generated. Egam1 transfectants promoted differentiation into cell types expressing Gata6, Gata4, Afp, or Plat, genes associated with emergence of the extra-embryonic endoderm lineages. On the other hand, Egam1 transfectants inhibited the expression of specific genes for the embryonic lineages, including Fgf5 (epiblast) and T (mesoderm), in addition to Cdx2, a specific gene for the extra-embryonic trophectoderm lineages. Changes in the percentage of cells recognizing by antibodies against specific marker proteins closely correlated with the expression patterns of their transcripts. Taken together, the results obtained in this study suggested that mouse ES cells expressing exogenous EGAM1 preferentially differentiate into extra-embryonic primitive endoderm lineages, rather than embryonic lineages or extra-embryonic trophectoderm lineages. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.