Human embryonic stem cells (hESCs) are offering a new therapeutic approach because of their unique ability to proliferate indefinitely in vitro and differentiate into multiple cell types. However, our understanding of the molecular mechanisms of pluripotency and self-renewal remain incomplete. To elucidate the key regulatory sequences and genes responsible for these cellular properties, we have determined potential enhancers and insulators in the genome of human ES cells and examined the dynamics of four key chromatin modifications (H3K4me1, H3K4me3, H3K27ac and H3K27me3) at both promoters and enhancers during the differentiation of these cells. We observe that most enhancers gain or lose H3K4me1 and H3K27ac during differentiation in a manner that correlates with expression of their potential target genes. By contrast, chromatin modifications at promoters remain stable and largely invariant during hESC differentiation, with the exception of a small number of promoters where a dynamic switch between acetylation and methylation at H3K27 marks the transition between activation and silencing of gene expression. Our results reveal more than 50,000 potential enhancers for early human development, and identify key genes that are involved in differentiation and maintenance of pluripotency in human ES cells. ChIP-Seq Analysis of SOX2 and NANOG in hESC H1 cells. 36 cycles of sequencing was done on the Illumina Genome Analyzer II platform.