PurposeEnteric nervous system (ENS) stem cells are present in embryonic and postnatal gut. Isolation of these cells would be valuable for treating a deficient or injured ENS. We aimed to study the effect of glial‐derived neurotrophic factor (GDNF) on growth and differentiation of ENS stem cells.Materials & MethodsEmbryonic rat intestine was digested and the cells grown in medium supplemented with EGF and bFGF. Neurosphere‐like bodies developed, dispersed, and plated in a collagen matrix at clonal density. These cultures were supplemented with EGF and bFGF, with and without GDNF (10 or 50 ng/ml). Colony number and size was recorded and their capacity to form new neurospheres tested.ResultsWhile control cultures developed colonies <0.5 mm in size, colonies in GDNF‐treated cultures were larger (>;2 mm). Secondary colonies arose from 0.04–0.4% of these cells, forming new neurospheres with neuroglial morphology.ConclusionsOur results demonstrate that neurosphere‐like bodies derived from rat embryonic gut are capable of self‐propagation and neuroglial differentiation, consistent with stem/progenitor cell behavior. In addition, we find that GDNF stimulates ENS stem cell proliferation to form large colonies that maintain the potential to differentiate into neural and glial phenotypes. This work was partially supported by a grant from CONACYT.