Neurogenin3 (NEUROG3) is required for endocrine lineage formation of the pancreas and intestine. Patients with NEUROG3 mutations are born with congenital malabsorptive diarrhea due to complete loss of enteroendocrine cells, whereas endocrine pancreas development varies in an allele-specific manner. These findings suggest a context-dependent requirement for NEUROG3 in pancreas versus intestine. We utilized human tissue differentiated from NEUROG3−/− pluripotent stem cells for functional analyses. Most disease-associated alleles had hypomorphic or null phenotype in both tissues, whereas the S171fsX68 mutation had reduced activity in the pancreas but largely null in the intestine. Biochemical studies revealed NEUROG3 variants have distinct molecular defects with altered protein stability, DNA binding, and gene transcription. Moreover, NEUROG3 was highly unstable in the intestinal epithelium, explaining the enhanced sensitivity of intestinal defects relative to the pancreas. These studies emphasize that studies of human mutations in the endogenous tissue context may be required to assess structure-function relationships. Display omitted •Human PSC-derived pancreas and intestinal endocrine cells to model NEUROG3 mutations•NEUROG3 mutations affect protein stability, dimerization, and DNA binding•NEUROG3 protein is less stable in intestinal epithelium than in the pancreas•Reduced protein stability in intestine enhances sensitivity to NEUROG3 mutations Zhang et al. use a human pluripotent stem cell-based system to study the impact of patient-derived mutations in NEUROG3 on the development of pancreatic and intestinal endocrine cells. This system recapitulates patient phenotypes, identifies hypomorphic alleles, and uncovers the mutations’ effects on dimerization, DNA binding, transcription, and protein stability.