Prospective isolation and characterization of progenitor cells is a paradigmatic strategy for studies of organ development. However, extraction of viable cells, fractionation of lineages, and in vitro analysis of progenitors from the fetal pancreas in experimental organisms like mice has proved challenging and has not yet been reported for human fetal pancreas. Here, we report isolation of pancreatic islet progenitor cells from fetal mice by FACS. Monoclonal antibodies that recognize cell-surface proteins on candidate stem cells in brain, skin, and other organs enabled separation of major pancreatic cell lineages and isolation of native pancreatic cells expressing neurogenin 3, an established marker of islet progenitors. New in vitro cell culture methods permitted isolated mouse islet progenitors to develop into hormone-expressing endocrine cells. Insulin-producing cells derived in vitro required or expressed factors that regulate fetal β cell differentiation; thus, the genetic programs normally controlling in vivo mouse islet development are similarly required in our system. Moreover, antibodies that recognize conserved orthologous cell-surface epitopes in human fetal pancreas allowed FACS-based enrichment of candidate islet progenitor cells expressing neurogenin 3. Our studies reveal previously undescribed strategies for prospective purification and analysis of pancreatic endocrine progenitor cells that should accelerate studies of islet development and replacement.