Multiple transcription factors have been shown to promote pancreatic β-cell differentiation, yet much less is known about negative regulators. Earlier epigenomic studies suggested that the transcriptional repressor REST could be a suppressor of endocrinogenesis in the embryonic pancreas. However, pancreatic Rest knockout mice failed to show abnormal numbers of endocrine cells, suggesting that REST is not a major regulator of endocrine differentiation. Using a different conditional allele that enables profound REST inactivation, we observed a marked increase in pancreatic endocrine cell formation. REST inhibition also promoted endocrinogenesis in zebrafish and mouse early postnatal ducts and induced β-cell-specific genes in human adult duct-derived organoids. We also defined genomic sites that are bound and repressed by REST in the embryonic pancreas. Our findings show that REST-dependent inhibition ensures a balanced production of endocrine cells from embryonic pancreatic progenitors.