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Summary: Transcriptional regulatory mechanisms of lineage priming in embryonic development are largely uncharacterized because of the difficulty of isolating transient progenitor populations. Directed differentiation of human pluripotent stem cells (hPSCs) combined with gene editing provides a powerful system to define precise temporal gene requirements for progressive chromatin changes during cell fate transitions. Here, we map the dynamic chromatin landscape associated with sequential stages of pancreatic differentiation from hPSCs. Our analysis of chromatin accessibility dynamics led us to uncover a requirement for FOXA2, known as a pioneer factor, in human pancreas specification not previously shown from mouse knockout studies. FOXA2 knockout hPSCs formed reduced numbers of pancreatic progenitors accompanied by impaired recruitment of GATA6 to pancreatic enhancers. Furthermore, FOXA2 is required for proper chromatin remodeling and H3K4me1 deposition during enhancer priming. This work highlights the power of combining hPSC differentiation, genome editing, and computational genomics for discovering transcriptional mechanisms during development. : Lee et al. use ATAC-seq to identify key transcriptional factors involved in human pancreatic differentiation. FOXA2 knockout human pluripotent stem cells showed impaired differentiation to pancreatic progenitors, a phenotype not observed in Foxa2 conditional knockout mice. Furthermore, FOXA2 is required for proper chromatin remodeling and H3K4me1 deposition during enhancer priming. Keywords: human pluripotent stem cells, hPSCs, human embryonic stem cells, hESCs, pancreatic progenitors, ATAC-seq and chromatin accessibility, FOXA2, FOXA1, enhancer priming and activation, nucleosome remodeling, PDX1, GATA6