Defining the precise regionalization of specified definitive endoderm progenitors is critical for understanding the mechanisms underlying the generation and regeneration of respiratory and digestive organs,yet the patterning of endoderm progenitors remains unresolved,particularly in humans.We performed single-cell RNA sequencing on endoderm cells during the early somitogenesis stages in mice and humans.We developed molecular criteria to define four major endoderm regions (foregut,lip of anterior intestinal portal,midgut,and hindgut) and their developmental pathways.We identified the cell subpopulations in each region and their spatial distributions and characterized key molecular features along the body axes.Dorsal and ventral pancreatic progenitors appear to originate from the midgut population and follow distinct pathways to develop into an identical cell type.Finally,we described the generally conserved endoderm patterning in humans and clear differences in dorsal cell distribution between species.Our study comprehensively defines single-cell endoderm patterning and provides novel insights into the spatiotemporal process that drives establishment of early endoderm domains.