The inner cell mass of pre-implantation blastocyst stage embryos is a source of cells that can be cultured indefinitely in vitro as a self-renewing, pluripotent population. In this review, we discuss the hallmarks of pluripotent cells derived from murine and human embryos and compare signaling pathways and transcriptional networks required to maintain them in a stable, pluripotent state. Culture conditions required for maintenance of murine and human embryonic stem cells (ESCs) vary significantly, but numerous 'critical' factors have been identified as being important for ESC pluripotency. We will attempt to reconcile the literature in terms of what is critical, from a signal transduction perspective, for maintenance of pluripotency. Finally, we consider recent findings describing a new pluripotent population of cells derived from the mouse epiblast, which seem to be more closely related to hESCs than mESCs. This poses some interesting questions as to the developmental equivalence of hESCs and suggests how we need to re-evaluate how we think of hESCs in the future.