The enteric nervous system (ENS) is organized into neural circuits within the gastrointestinal wall where it controls the peristaltic movements, secretion, and blood flow. Although proper gut function relies on the complex neuronal composition of the ENS, little is known about the transcriptional networks that regulate the diversification into different classes of enteric neurons and glia during development. Here we redefine the role of Ascl1 (Mash1), one of the few regulatory transcription factors described during ENS development. We show that enteric glia and all enteric neuronal subtypes appear to be derived from Ascl1-expressing progenitor cells. In the gut ofAscl1^−/−mutant mice, neurogenesis is delayed and reduced, and posterior gliogenesis impaired. The ratio of neurons expressing Calbindin, TH, and VIP is selectively decreased while, for instance, 5-HT⁺neurons, which previously were believed to be Ascl1-dependent, are formed in normal numbers. Essentially the same differentiation defects are observed inAscl1^KINgn2transgenic mutants, where the proneural activity of Ngn2 replaces Ascl1, demonstrating that Ascl1 is required for the acquisition of specific enteric neuronal subtype features independent of its role in neurogenesis. In this study, we provide novel insights into the expression and function of Ascl1 in the differentiation process of specific neuronal subtypes during ENS development.SIGNIFICANCE STATEMENTThe molecular mechanisms underlying the generation of different neuronal subtypes during development of the enteric nervous system are poorly understood despite its pivotal function in gut motility and involvement in gastrointestinal pathology. This report identifies novel roles for the transcription factor Ascl1 in enteric gliogenesis and neurogenesis. Moreover, independent of its proneurogenic activity, Ascl1 is required for the normal expression of specific enteric neuronal subtype characteristics. Distinct enteric neuronal subtypes are formed in a temporally defined order, and we observe that the early-born 5-HT⁺neurons are generated inAscl1^−/−mutants, despite the delayed neurogenesis. Enteric nervous system progenitor cells may therefore possess strong intrinsic control over their specification at the initial waves of neurogenesis.