Delineating key factors that regulate early pancreas development is crucial to our long-term pursuit of understanding disease mechanisms and developing human pluripotent stem cell (hPSC) based β-cell replacement therapies for diabetes. However, a complete landscape of signaling cues and transcription factors required for pancreas specification remains unclear. Using hPSC directed differentiation and CRISPR/Cas-mediated genome editing, recently published work from the Huangfu lab has identified critical new requirements for RFX6, GATA6 and GATA4 (genes known to be associated with neonatal and adult-onset diabetes) during human pancreatic differentiation. In unpublished work, we have further uncovered a novel, dose-dependent requirement for FOXA2, a gene associated with diabetes and hyperinsulinism, in pancreatic differentiation. Here in this collaboration between the Huangfu, Leslie and Pe’er labs, we will undertake complementary genetic, genomic and computational approaches and utilize hPSC differentiation to dissect human pancreatic development. We will utilize genetic approach to create precise hPSC disease models and interrogate complex genetic interactions underlying disease phenotypes, and employ genomic approaches including ChIP-seq and ATAC-seq analyses and cutting-edge single-cell transcriptomics to understand regulators of human pancreatic development and β cell function. Our findings will enhance the understanding of human pancreatic development and disease, and facilitate the development of improved hPSC directed differentiation protocols for the generation of functional β cells for disease study and treatment.