Self-renewal of embryonic stem cells (ESCs) is maintained by a complex regulatory mechanism involving transcription factors Oct3/4 (Pou5f1), Nanog and Sox2. Here, we report that Klf5, a Zn-finger transcription factor of the Kruppel-like family, is involved in ESC self-renewal. Klf5 is expressed in mouse ESCs, blastocysts and primordial germ cells, and its knockdown by RNA interference alters the molecular phenotype of ESCs, thereby preventing their correct differentiation. The ability of Klf5 to maintain ESCs in the undifferentiated state is supported by the finding that differentiation of ESCs is prevented when Klf5 is constitutively expressed. Maintenance of the undifferentiated state by Klf5 is, at least in part, due to the control of Nanog and Oct3/4 transcription, because Klf5 directly binds to the promoters of these genes and regulates their transcription.