Purpose <p>Intestinal neuronal dysplasia (IND) features abnormal enteric nervous system (ENS) development and symptom overlap with Hirschsprung’s disease, yet fetal-stage mechanisms remain unclear. We established a <i>Sox10</i>-Venus⁺/<i>Ncx</i>^{<i>⁻/⁻</i>} mouse model of IND, enabling fluorescent labeling of enteric neural crest cells (ENCCs) to test the hypothesis whether neuronal/glial differentiation abnormalities arise during early fetal stages.</p> Methods <p>ENCCs were isolated from embryonic day 13.5 (E13.5) fetal gut of <i>Sox10</i>-Venus⁺/<i>Ncx</i>^{<i>⁻/⁻</i>} (n = 6) and <i>Sox10</i>-Venus⁺/<i>Ncx</i>^{<i>⁺/⁺</i>} (n = 6) embryos, dissociated, and cultured under non-adherent conditions for 14&#xa0;days to generate neurospheres. Neurosphere diameter and the proportion of SOX10⁺ cells were measured using epifluorescence microscopy. Differentiation on day 14 was assessed by immunofluorescence for TUJ1 (neuronal) and S100β (S100B, glial) markers.</p> Results <p><i>Ncx</i>^{<i>⁻/⁻</i>} neurospheres were significantly larger than controls on days 5 and 10, with no significant difference on day 14. The proportion of SOX10⁺ cells remained higher through day 10. On day 14, <i>Ncx</i>^{<i>⁻/⁻</i>} neurospheres exhibited a lower proportion of TUJ1⁺ cells, preserved S100β⁺ cell proportions, and disrupted spheroid organization with heterogeneous marker distribution.</p> Conclusion <p>These findings demonstrate that <i>Ncx</i> deficiency leads to abnormal ENS development beginning during the fetal period, providing a mechanistic basis for postnatal hyperganglionosis and validating this model for studying IND pathogenesis.</p>