Stem cells are defined by the simultaneous possession of the seemingly incongruent properties of self-renewal and multi-lineage differentiation potential. To maintain a stem cell population, these opposing forces must be balanced. Transcription factors that function to direct pluripotent cell identity are not all equally distributed throughout the pluripotent cell population. While Oct4 levels are relatively homogeneous, other transcription factors, such as Nanog, are more heterogeneously expressed. Moreover, Oct4 positive cells fluctuate between states of high Nanog expression associated with a high probability of self-renewal and low Nanog expression associated with an increased propensity to differentiate. As embryonic stem (ES) cells transit to the more developmentally advanced epiblast stem cell (EpiSC) state, the levels of pluripotency transcription factors are modulated. Such modulations are blunted in cells that overexpress Nanog and this may underlie the resistance of Nanog-overexpressing cells to transit to an EpiSC state. Interestingly, increasing the levels of Nanog in EpiSC can facilitate reversion to the ES cell state. Together these observations suggest that Nanog lies close to the top of the hierarchy of pluripotent transcription factor regulation.