Developmental neurotoxicity (DNT) is increasingly recognized as a public health concern due to evidence linking environmental exposures to neurodevelopmental disorders. Traditional in vivo approaches are resource-intensive, highlighting the need for alternative in vitro models. In this context, 3D neurosphere cultures derived from neural stem cells (NSCs) provide a promising platform to evaluate key neurodevelopmental processes and their disruption by toxicants. This study aimed to develop and characterize a 3D neurosphere model of cortical differentiation and assess its response to chronic exposure to the pesticide chlorpyrifos (CPF) and a representative mixture of per- and polyfluoroalkyl substances (PFAS). After 21 days of spontaneous differentiation, the model expressed genes corresponding to multiple neurodevelopmental endpoints—neurogenesis, gliogenesis, synaptogenesis, and neuronal signaling—as confirmed by means of qPCR and flow cytometry. Additional phenotypic endpoints included cell proliferation, neurosphere size, radial migration, and cytotoxicity (XTT assay). To assess the model’s sensitivity to environmental toxicants, neurospheres were chronically exposed from Day 0 of differentiation, targeting a critical developmental window. The toxicants tested were CPF (125 μM and 250 μM) and a PFAS mixture at concentrations relevant to human serum exposure. CPF significantly reduced cell viability, inhibited sphere growth and radial migration, and disrupted gene expression across neurodevelopmental pathways, including the downregulation of genes involved in neuronal and glial differentiation and synaptogenesis, while upregulating early neurogenesis and signaling markers, particularly NTRK1. Furthermore, cytometry revealed CPF-induced alterations in the cell cycle profile and increased expression of proliferation markers. PFAS exposure, though not overtly cytotoxic, caused significant transcriptional disturbances in glial and neuronal genes, suggesting early interference with developmental programming even at non-cytotoxic, environmentally relevant levels. This neurosphere model recapitulates key stages of brain development and enables integrated molecular and functional DNT analysis. Its responsiveness to CPF and PFAS supports its relevance as an in vitro platform for chemical safety assessment.