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Pluripotency and the capability for self-renewal are essential characteristics of human embryonic stem cells (hESCs), which hold great potential as a cellular source for tissue replacement. Short cell cycle (15-16 h) compared to somatic cells is another property of hESCs. Efficient synchronization of hESCs at different cell cycle stages is important to elucidate the mechanistic link between cell cycle regulation and cell fate decision. This protocol describes how to establish synchronization of hESCs at different cell cycle stages.