Human pluripotent stem cell (hPSC) differentiation typically yields heterogeneous populations. Knowledge of signals controlling embryonic lineage bifurcations could efficiently yield desired cell types through exclusion of alternate fates. Therefore, we revisited signals driving induction and anterior-posterior patterning of definitive endoderm to generate a coherent roadmap for endoderm differentiation. With striking temporal dynamics, BMP and Wnt initially specified anterior primitive streak (progenitor to endoderm), yet, 24 hr later, suppressed endoderm and induced mesoderm. At lineage bifurcations, cross-repressive signals separated mutually exclusive fates; TGF-β and BMP/MAPK respectively induced pancreas versus liver from endoderm by suppressing the alternate lineage. We systematically blockaded alternate fates throughout multiple consecutive bifurcations, thereby efficiently differentiating multiple hPSC lines exclusively into endoderm and its derivatives. Comprehensive transcriptional and chromatin mapping of highly pure endodermal populations revealed that endodermal enhancers existed in a surprising diversity of “pre-enhancer” states before activation, reflecting the establishment of a permissive chromatin landscape as a prelude to differentiation. Display omitted •Signaling roadmap for four successive lineage bifurcations during endoderm development•Highly efficient (94%) endodermal differentiation of nine diverse hPSC lines•Global mapping of endoderm enhancer dynamics across five distinct lineage transitions•H2AZ marks an endoderm “pre-enhancer” state prior to activation Loh et al. describe an efficient protocol for the endodermal differentiation of human pluripotent stem cells by directing signals controlling lineage bifurcations, allowing precise analysis of the chromatin status of enhancers during the differentiation process.