Organophosphate pesticides irreversibly and semi‐permanently inhibit acetylcholinesterase, the enzyme responsible for the degradation of the neurotransmitter acetylcholine. Therefore, inhibition of acetylcholinesterase by organophosphate pesticides causes hyperstimulation of the cholinergic nervous system affecting the development and function of neurons in the CNS and PNS. This study seeks to understand the acute effects of organophosphate pesticides, including chlorpyrifos, on differentiating PC‐12 cells, and the effects of long‐term exposure on differentiated PC‐12 cell viability and susceptibility to neurotoxic agents. A dose‐dependent increase in cell death was observed for PC‐12 cells exposed to chlorpyrifos during differentiation. Additionally, results indicated that differentiating PC‐12 cells were most sensitive to chlorpyrifos at mid to late stages of differentiation when the neurons were beginning to form synapses. At earlier stages, chlorpyrifos had little effect on cell survival. Long‐term exposure to a sub‐lethal concentration of chlorpyrifos caused a decrease in neuronal viability, and influenced their susceptibility to H2O2‐induced oxidative damage and Aβ42‐induced damage. Together these data demonstrate that organophosphate exposure adversely affects PC‐12 cell survival and long‐term exposure alters their susceptibility to agents involved in neurodegenerative disease.