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Recently, it has been suggested that p27Kip1, the cell cycle regulatory protein, plays a pivotal role in the progression of normal differentiation in murine embryonic stem (mES) cells. In the current study, we investigated the role of p27Kip1 in the regulation of differentiation and apoptotic induction using Western blotting, quantitative real-time RT-PCR, and small interfering RNA (siRNA) assays and confocal laser scanning microscopic analysis of H9 human ES (hES) cells and H9-derived embryoid bodies (EBs)grown for 10 (EB10) and 20 days (EB20). Our results demonstrate that the proteins p27Kip1 and cyclin D3 are strongly associated with cellular differentiation, and, for the first time, show that up-regulation of p27Kip1 protects hES cells from inducing differentiation-associated and caspase 3-dependent apoptosis.