Embryonic stem cells (ESC) have been isolated from pregastrulation mammalian embryos. The maintenance of their pluripotency and ability to self-renew has been shown to be governed by the transcription factors Oct4 (Pou5f1) and Nanog. Oct4 appears to control cell-fate decisions of ESC in vitro and the choice between embryonic and trophectoderm cell fates in vivo. In non-mammalian vertebrates, the existence and functions of these factors are still under debate, although the identification of the zebrafish pou2 (spg; pou5f1) and Xenopus Pou91 (XlPou91) genes, which have important roles in maintaining uncommitted putative stem cell populations during early development, has suggested that these factors have common functions in all vertebrates. Using chicken ESC (cESC), which display similar properties of pluripotency and long-term self-renewal to mammalian ESC, we demonstrated the existence of an avian homologue of Oct4 that we call chicken PouV (cPouV). We established that cPouV and the chicken Nanog gene are required for the maintenance of pluripotency and self-renewal of cESC. These findings show that the mechanisms by which Oct4 and Nanog regulate pluripotency and self-renewal are not exclusive to mammals.