Differentiation of enteric neural stem cells into several appropriate neural phenotypes is crucial while considering transplantation as a cellular therapy to treat enteric neuropathies. We describe the formation of tissue engineered innervated sheets, where intestinal smooth muscle and enteric neuronal progenitor cells are brought into close association in extracellular matrix (ECM) based microenvironments. Uniaxial alignment of constituent smooth muscle cells was achieved by substrate microtopography. The smooth muscle component of the tissue engineered sheets maintained a contractile phenotype irrespective of the ECM composition, and generated equivalent contractions in response to potassium chloride stimulation, similar to native intestinal tissue. We provided enteric neuronal progenitor cells with permissive ECM-based compositional and viscoelastic cues to generate excitatory and inhibitory neuronal subtypes. In the presence of the smooth muscle cells, the enteric neuronal progenitor cells differentiated to functionally innervate the smooth muscle. The differentiation of specific neuronal subtypes was influenced by the ECM microenvironment, namely combinations of collagen I, collagen IV, laminin and/or heparan sulfate. The physiology of differentiated neurons within tissue engineered sheets was evaluated. Sheets with composite collagen and laminin had the most similar patterns of Acetylcholine-induced contraction to native intestinal tissue, corresponding to an increased protein expression of choline acetyltransferase. An enriched nitrergic neuronal population, evidenced by an increased expression of neuronal nitric oxide synthase, was obtained in tissue engineered sheets that included collagen IV. These sheets had a significantly increased magnitude of electrical field stimulated relaxation, sensitive maximally to nitric oxide synthase inhibition. Tissue engineered sheets containing laminin and/or heparan sulfate had a balanced expression of contractile and relaxant motor neurons. Our studies demonstrated that neuronal subtype was modulated by varying ECM composition. This observation could be utilized to derive enriched populations of specific enteric neurons in vitro prior to transplantation.