Gut motility disorders can result from an absent, damaged, or dysfunctional enteric nervous system (ENS). Cell therapy is an exciting prospect to treat these enteric neuropathies and restore gut motility. Previous studies have examined a variety of sources of stem/progenitor cells, but the ability of different sources of cells to generate enteric neurons has not been directly compared. It is important to identify the source of stem/progenitor cells that is best at colonizing the bowel and generating neurons following transplantation. The aim of this study was to compare the ability of central nervous system (CNS) progenitors and ENS progenitors to colonize the colon and differentiate into neurons. Genetically labeled CNS- and ENS-derived progenitors were cocultured with aneural explants of embryonic mouse colon for 1 or 2.5 wk to assess their migratory, proliferative, and differentiation capacities, and survival, in the embryonic gut environment. Both progenitor cell populations were transplanted in the postnatal colon of mice in vivo for 4 wk before they were analyzed for migration and differentiation using immunohistochemistry. ENS-derived progenitors migrated further than CNS-derived cells in both embryonic and postnatal gut environments. ENS-derived progenitors also gave rise to more neurons than their CNS-derived counterparts. Furthermore, neurons derived from ENS progenitors clustered together in ganglia, whereas CNS-derived neurons were mostly solitary. We conclude that, within the gut environment, ENS-derived progenitors show superior migration, proliferation, and neuronal differentiation compared with CNS progenitors., (Copyright © 2014 the American Physiological Society.)