Activation of Nrf2/ARE signaling pathway occurs ubiquitously in most cell types upon induction of oxidative stress. Rotenone, an inhibitor of mitochondrial complex I, can be used to trigger oxidative stress, stimulate the activation of Nrf2 pathway in neuronal and astrocytic cells and assess neurotoxicity. We have previously demonstrated that an acute treatment with rotenone can induce Nrf2 activation, which leads to astrocyte activation and dopaminergic (DA) neuronal cell death in a mixed neuronal/astrocytic cell model derived from human induced pluripotent stem cells (hiPSCs). In this study, we characterized the effects of a repeated dose treatment with rotenone (14 days) on hiPSC-derived neural stem cells (NSCs) undergoing differentiation, assessing the expression and the activation of the Nrf2 pathway. Our results show that Nrf2 signaling increases during NSC differentiation. Moreover, we observed that rotenone treatment induced a progressive activation of Nrf2 signaling together with a induction of astrocyte reactivity, a reduction of neurite length leading to neuronal cell death, in particular of DA neurons. Altogether these data indicate that hiPSC-NSC models are relevant test systems for the evaluation of Nrf2 pathway activation upon induced oxidative stress, allowing further understanding of the molecular mechanisms underlying exposure to (developmental) neurotoxicants.