The transcription factor Oct‐4 is a marker of pluripotency in mouse and human embryonic stem (ES) cells. Previous studies using a tetracycline‐regulated Oct‐4 transgene in the ZHBTc4 cell line demonstrated that downregulation of Oct‐4 expression induced dedifferentiation into trophoblast, a lineage mouse ES cells do not normally generate. We found that transfection of Oct‐4‐specific short interfering RNA significantly reduced expression and functional activity of Oct‐4 in mouse and human ES cells, enabling its role to be compared in both cell types. In mouse ES cells, Oct‐4 knockdown produced a pattern of morphological differentiation and increase in expression of the trophoblast‐associated transcription factor Cdx2, similar to that triggered by suppressing the Oct‐4 transgene in the ZHBTc4 cell line. In addition, downregulation of Oct‐4 was accompanied by increased expression of the endoderm‐associated genes Gata6 and α‐fetoprotein, and a gene trap associated with primitive liver/yolk sac differentiation. In human ES cells, Oct‐4 knockdown also induced morphological differentiation coincident with the upregulation of Gata6. The induction of Cdx2 and other trophoblast‐associated genes, however, was dependent on the culture conditions. These results establish the general requirement for Oct‐4 in maintaining pluripotency in ES cells. Moreover, the upregulation of endoderm‐associated markers in both mouse and human ES cells points to overlap between development of trophoblast and endoderm differentiation.