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Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) hold great promise in regenerative medicine. Realizing this potential requires a thorough understanding of the genetic programs regulating their pluripotency and lineage commitment. Recent studies have generated a wealth of information regarding the transcriptional circuitry underlying self-renewal and lineage commitment of ES cells. Here we review these studies, focusing on a type of cis-regulatory sequences called enhancers and their potential roles in pluripotency and cell fate determination. We also discuss how such information can help ES cell research and regenerative medicine, together with the challenges to understand the functions and regulation of these elements.