To characterize the in vivo dynamics of enhancer usage during development, Axel Visel and colleagues generated genome-wide maps of the enhancers active in mouse forebrain, heart and liver (Cell 155, 1521–1531, 2013). The authors profiled acetylation at histone H3 lysine 27 (H3K27ac), a histone modification associated with active enhancers and transcription start sites (TSSs), via chromatin immunoprecipitation and sequencing (ChIP-seq) of mouse tissue collected at seven different stages of pre- and postnatal development. Their analyses identified 105,394 H3K27ac-enriched regions, comprising 16,225 TSS-proximal regions and 89,169 TSS-distal regions that represent candidate enhancers, and characterized patterns of dynamic enhancer activity spatially and temporally across development. Almost 50% of candidate enhancers were predicted to be active in only one of the three tissues, and just 3% of candidate enhancers were marked by H3K27ac enrichment in all tissues and at all stages. To experimentally validate enhancer predictions, the authors used a transgenic mouse enhancer reporter assay to test candidate forebrain enhancers, finding that 23 of 32 (72%) candidate enhancers tested in transgenic mice drove expression patterns in vivo as predicted by H3K27ac marks.