The pluripotency and self-renewal capacity of embryonic stem (ES) cells is regulated by several transcription factors. Here, we show that the ETS-related transcription factors Etv4 and Etv5 (Etv4/5) are specifically expressed in undifferentiated ES cells, and suppression of Oct3/4 results in down-regulation of Etv4/5. Simultaneous deletion of Etv4 and Etv5( Etv4/5doubleknock-out(dKO)) in ES cells resulted in a flat, epithelial cell-like appearance, whereas the morphology changed into compact colonies in a 2i medium (containing two inhibitors for GSK3 and MEK/ERK). Expression levels of self-renewal marker genes, including Oct3/4 and Nanog, were similar between wild-type and dKO ES cells, whereas proliferation of Etv4/5 dKO ES cells was decreased with overexpression of cyclin-dependent kinase inhibitors (p16/p19, p15, and p57). A differentiation assay revealed that the embryoid bodies derived from Etv4/5 dKO ES cells were smaller than the control, and expression of ectoderm marker genes, including Fgf5, Sox1, and Pax3, was not induced in dKO-derived embryoid bodies. Microarray analysis demonstrated that stem cell-related genes, including Tcf15, Gbx2, Lrh1, Zic3, and Baf60c, were significantly repressed in Etv4/5 dKO ES cells. The artificial expression of Etv4 and/or Etv5 in Etv4/5 dKO ES cells induced re-expression of Tcf15 and Gbx2. These results indicate that Etv4 and Etv5, potentially through regulation of Gbx2 and Tcf15, are involved in the ES cell proliferation and induction of differentiation-associated genes in ES cells.