With the increase in fetal surgeries, the effect of maternal anesthesia on progeny has attracted much attention. Our previous studies have demonstrated that 3.5% sevoflurane maternal exposure resulted in over-activated autophagy and cognitive impairment in the offspring. The autophagy activation resulted in increased apoptosis and decreased proliferation. However, the effects of sevoflurane on neural stem cell (NSC) differentiation is unclear. There is evidence that autophagy might participate in anesthesia-induced NSC differentiation. Firstly, we examined the effects of sevoflurane on NSC differentiation and explored possible mechanisms. Then, we investigated whether autophagy was related to differentiation. On gestational day 14 (G14), rats were exposed to 2% or 3.5% sevoflurane for 2 h, then markers of neurons and astrocytes, and the FOXO3 expression was measured in fetal brains 48 h later. The differentiation of NSCs was detected after autophagy inhibition by 3-MA. Changes in NSC differentiation, autophagy level, and FOXO3 were examined after administration of lithium chloride. After 3.5% sevoflurane exposure, the expressions of β-Tubulin III, NeuN, SYP, GFAP and FOXO3 increased. Autophagy inhibition alleviates improper NSC differentiation. Lithium chloride attenuated FOXO3 and autophagy activation, ameliorated NSC differentiation and the decline of Nestin expression. Our results demonstrated that maternal exposure to 3.5% sevoflurane for 2 h during the mid-trimester induced NSC differentiation in the fetal brain through the activation of FOXO3. Autophagy inhibitor or lithium chloride reversed the improper differentiation of NSCs.