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SUMMARY Stem-cell differentiation to desired lineages requires navigating alternating developmental paths often leading to unwanted cell-types. Hence comprehensive developmental roadmaps are crucial to channel stem-cell differentiation towards desired fates. To this end, here we map bifurcating lineage choices leading from pluripotency to twelve human mesodermal lineages, including bone, muscle and heart. We defined the extrinsic signals controlling each binary lineage decision, enabling us to logically block differentiation towards unwanted fates and rapidly steer pluripotent stem cells towards 80–99% pure human mesodermal lineages at most branchpoints. This strategy enabled the generation of human bone and heart progenitors that could engraft in respective in vivo models. Mapping stepwise chromatin and single-cell gene expression changes in mesoderm development uncovered somite segmentation, a previously-unobservable human embryonic event transiently marked by HOPX expression. Collectively this roadmap enables navigation of mesodermal development to produce transplantable human tissue progenitors and uncover developmental processes.

Here we describe Protein Interaction Quantitation (PIQ), a computational method that models the magnitude and shape of genome-wide DNase profiles to facilitate the identification of transcription factor (TF) binding sites. Through the use of machine learning techniques, PIQ identified binding sites for >700 TFs from one DNase-seq experiment with accuracy comparable to ChIP-seq for motif-associated TFs (median AUC=0.93 across 303 TFs). We applied PIQ to analyze DNase-seq data from mouse embryonic stem cells differentiating into pre-pancreatic and intestinal endoderm. We identified (n=120) and experimentally validated eight ‘pioneer’ TF families that dynamically open chromatin, enabling other TFs to bind to adjacent DNA. Four pioneer TF families only open chromatin in one direction from their motifs. Furthermore, we identified a class of ‘settler’ TFs whose genomic binding is principally governed by proximity to open chromatin. Our results support a model of hierarchical TF binding in which directional and non-directional pioneer activity shapes the chromatin landscape for population by settler TFs.