Combinatorial BMP4 and activin direct the choice between alternate routes to endoderm in a stem cell model of human gastrulation

Lineage specification requires accurate interpretation of multiple signaling cues. However, how combinatorial signaling histories influence fate outcomes remains unclear. We combined single-cell transcriptomics, live-cell imaging, and mathematical modeling to explore how activin and bone morphogenetic protein 4 (BMP4) guide fate specification during human gastrulation. We see that these signals interact both synergistically and antagonistically to drive fate decisions. We find that definitive endoderm arises from lineage convergence: a direct route from pluripotency and an indirect route via a mesoderm progenitor state. Cells pass through temporal windows of signaling competency, and the relative concentration of activin and BMP4 dictates the trajectory choice. The efficiency between routes is underpinned by a dual role of BMP4 in inducing mesoderm genes while promoting pluripotency exit. This work underscores that the combination of signals a cell is exposed to not only directs its final fate but also the developmental route taken, suggesting lineage convergence enhances robustness in fate specification. • Human definitive endoderm is formed by multiple developmental routes • Endoderm cells derived from alternate origins have the same forward potential • Activin and BMP4 signaling determine the choice between alternate developmental routes • Dual BMP4 role affects the efficiency of alternate developmental routes to the endoderm Inge et al . show that human endoderm originates from two converging developmental routes with distinct dynamics and efficiencies yet similar developmental potential. Combinatorial activin and BMP4 signaling governs the choice between these routes, showing that signaling inputs not only determine a cell’s fate but also influence the developmental path taken.