Differentiating neuronal cells derived from human umbilical cord blood stem cells have been used as an in vitro tool for the assessment of developmental neurotoxicity of monocrotophos (MCP), an organophosphate pesticide. The differentiating cells were exposed to MCP during the different stages of maturation, viz., days 2, 4, and 8, and changes in the makers of cell proliferation, neuronal differentiation, neuronal injuries, and receptors were studied. We found significant upregulation in the different MAPKs, apoptosis, and neurogenesis markers and downregulation in the cell proliferation markers during neuronal differentiation. We further identified significant upregulation in the expression of different MAPKs and proteins involved in oxidative stress, apoptosis, and calpain pathways in the mid-differentiating cells exposed to MCP. The upregulated levels of these proteins seem to be the main cause of alteration during the differentiation process towards apoptosis as a fine-tune of pro-apoptotic and anti-apoptotic proteins are desirable for the process of differentiation without apoptosis. The decreased acetylcholinesterase activity, dopaminergic, and cholinergic receptors and increased acetylcholine levels in the differentiating neuronal cells indicate the vulnerability of these cells towards MCP-induced neurotoxicity. Our data confirms that differentiating neuronal cells derived from human umbilical cord stem cells could be used as a powerful tool to assess the developmental neurotoxicity in human beings.