Isolating adult stem cells from human stomach epithelia has proven challenging due to a lack of surface markers that are amenable to antibody-based sorting approaches. Here, through comparative expression profiling of mouse Lgr5+ adult stem cells along the GI tract, we identify novel pylorus-specific stem cell markers, including the membrane protein Aqp5. FACS-based isolation of endogenous AQP5+ cells from healthy human pyloric stomach revealed their stem cell identity in organoid assays. Using new Aqp5-driven CreERT2 mouse models to selectively target conditional mutations to the pyloric stem cell compartment in vivo, we establish pyloric stem cells as a major source of Wnt-driven invasive gastric cancer. The new stem cell markers and mouse models described here will be an invaluable resource for deciphering early gastric cancer formation and for isolating and characterizing human stomach stem cells as a prerequisite to potentially exploiting their regenerative medicine potential in the clinic. Overall design: Stem and differentiated popualtions from various mouse gastrointestinal tissues were isolated by FACS for RNA extraction and hybridization on Affymetrix microarrays. We sought to identify genes specifically enriched in pyloric stem cells relative to the other gastrointestinal populations.