Enteric neural crest cells (ENCCs) are progenitor cells in the developing gut that give rise to the enteric nervous system (ENS). Mechanisms that regulate the migration, proliferation, and differentiation of these cells are crucial to the proper morphogenesis of the intestinal neural network during embryogenesis. Despite the relatively low population of these cells within the gut tissue, methods for isolation and in vitro culture have been established to enrich this cell population and enable investigations into molecular pathways that govern ENS development. Here, we describe a method to generate ENCC-derived neurospheres from the mouse embryonic gut at embryonic day (E) 13.5 by neural selective media enrichment.