Morphogen gradients pattern the endoderm and specify liver and pancreatic progenitors in vivo. However, if specified organ progenitors can be identified and isolated during human pluripotent stem cell (hPSC) differentiation is unknown. Here, we report the identification of two novel surface markers, CD177/NB1 glycoprotein and inducible T cell co-stimulatory ligand CD275/ICOSL, that isolate specified organ progenitors from seemingly homogenous endoderm differentiations in vitro. These markers allow assessing anterior definitive endoderm (ADE) patterning and specification in human revealing different morphogen requirements and induction efficiencies for the generation of specified pancreatic and liver progenitors using known and novel differentiation paradigms. Furthermore, molecular profiling and characterisation of CD177 + and CD275 + ADE subpopulations identified differential expression of signalling components and inverse activation of canonical and non-canonical WNT signalling. This signalling milieu specifies CD275 + ADE progenitors towards the liver fate. In contrast, CD177 + ADE progenitors express and synthesize the secreted WNT, NODAL and BMP antagonist CERBERUS1 and are specified towards the pancreatic fate. Strikingly, isolated CD177 + ADE progenitors differentiate more homogenously into pancreatic progenitors as well as into functionally, more mature and glucose-responsive β-like cells in vitro, when compared to bulk endoderm differentiations. Overall, the identification of novel surface markers allowed us to isolate, monitor and understand human organ progenitor formation for the improved differentiation of β-like cells from hPSC.