Methods for differentiating human pluripotent stem cells to pancreatic and liver lineages in vitro have been limited by the inability to identify and isolate distinct endodermal subpopulations specific to these two organs. Here we report that pancreatic and hepatic progenitors can be isolated using the surface markers CD177/NB1 glycoprotein and inducible T-cell costimulatory ligand CD275/ICOSL, respectively, from seemingly homogeneous definitive endoderm derived from human pluripotent stem cells. Anterior definitive endoderm (ADE) subpopulations identified by CD177 and CD275 show inverse activation of canonical and noncanonical WNT signaling. CD177+ ADE expresses and synthesizes the secreted WNT, NODAL and BMP antagonist CERBERUS1 and is specified toward the pancreatic fate. CD275+ ADE receives canonical Wnt signaling and is specified toward the liver fate. Isolated CD177+ ADE differentiates more homogeneously into pancreatic progenitors and into more functionally mature and glucose-responsive β-like cells in vitro compared with cells from unsorted differentiation cultures.