A unique chromatin signature identifies two classes of regulatory elements in human embryonic stem cells (hESCs). These elements are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. The first class, termed class I elements, is associated with active enhancers and is characterized by H3K27ac, overlap with previously characterized hESC enhancers, and proximity to genes expressed in hESCs and the epiblast. The second class, termed 'poised enhancers' (class II elements), is distinguished by the absence of H3K27ac, enrichment of H3K27me3, and association with genes inactive in hESCs but involved in early embryogenesis. During differentiation of hESCs to neuroepithelium, a subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers direct cell-type and stage-specific expression, even in the absence of sequence conservation. The study identifies over 2,000 putative regulatory sequences, providing a valuable resource for studying early human development. The findings suggest that early developmental enhancers are epigenetically pre-marked in hESCs, with H3K27me3 playing an unappreciated role at distal regulatory elements. The study also reveals that class II elements are poised enhancers that become active during development, acquiring an active chromatin signature upon differentiation. The results highlight the importance of chromatin signatures in defining enhancer function and provide insights into the regulation of gene expression during early development.A unique chromatin signature identifies two classes of regulatory elements in human embryonic stem cells (hESCs). These elements are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. The first class, termed class I elements, is associated with active enhancers and is characterized by H3K27ac, overlap with previously characterized hESC enhancers, and proximity to genes expressed in hESCs and the epiblast. The second class, termed 'poised enhancers' (class II elements), is distinguished by the absence of H3K27ac, enrichment of H3K27me3, and association with genes inactive in hESCs but involved in early embryogenesis. During differentiation of hESCs to neuroepithelium, a subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers direct cell-type and stage-specific expression, even in the absence of sequence conservation. The study identifies over 2,000 putative regulatory sequences, providing a valuable resource for studying early human development. The findings suggest that early developmental enhancers are epigenetically pre-marked in hESCs, with H3K27me3 playing an unappreciated role at distal regulatory elements. The study also reveals that class II elements are poised enhancers that become active during development, acquiring an active chromatin signature upon differentiation. The results highlight the importance of chromatin signatures in defining enhancer function and provide insights into the regulation of gene expression during early development.