Little is known about postnatal enteric nervous system (ENS) development, but some reports suggest that the postnatal bowel may contain neural stem cells. Therefore, we created an in vitro model of desegregation using an enzymatic and mechanical tissue technique. This approach yielded a group of cells from the small intestine of lactating and adult mice, which ex vivo attach to the culture dish; actively proliferate; and express nestin, vimentin, and the pro‐neural transcription factors neurogenin‐2 (ngn‐2), Sox‐10, and Mash‐1. In the conditions grown, double immunostains suggest that they differentiate into various cell types, particularly neurons, smooth muscle, and glia including 04 protein–positive cells. They also express the neurotrophic‐protein tyrosine kinase (Trk) receptors TrkA, TrkB, and TrkC; the low‐affinity neurotrophin receptor p75NTR; and the glial‐derived neurotrophic factor receptors (GFR)α‐1, GFRα‐2, and GFRα‐3. The neurons expressed several sensory and motor neurotransmitters present in the central and enteric nervous systems, including calcitonin gene‐related peptide, neuropeptideY, peptideYY, substance P, vasoactive intestinal polypeptide, and galanin; along with glia, these neurons formed elaborate intercellular connections. They also express c‐KIT, CD34, CD20, and CD45RO, suggesting they either have a hematogenous origin or may differentiate toward hematogenous lines. These findings suggest that these cells may be enteric neural stem cells (ENSCs); may normally be present in the small intestine; and may have the capacity to proliferate and differentiate into neurons, glia, and smooth muscle. Further identification and purification of intestinal ENSCs will provide a means to study the regulation of their differentiation and should give insight into the mechanisms involved in development and remodeling of the ENS. The possible therapeutic application of postnatal stem cells such as ENSCs needs to be evaluated, including their use for transplantation in the central nervous system.