The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27 Kip1 cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27 Kip1 in hESC lead to a G 1 phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27 Kip1 caused an elongated/scatter cell-like phenotype involving upregulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27 Kip1 protein occupies the Twist1 gene promoter and manipulation of p27 Kip1 by gain and loss of function is associated with Twist gene expression changes. These results define p27 Kip1 expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27 Kip1 in controlling an epithelial to mesenchymal transition (EMT) in hESC. © 2011 Landes Bioscience.