The aim of this study was to investigate the effects of hypoxia conditioned medium (HCM) of cerebral cortex cells on the differentiation of neural stem cells (NSCs) and to clarify the signal transduction mechanism. The cerebral cortex cells from newborn SD rats were primarily cultured for 5 d, and then the cells were cultured in environments of 4% O2, 1% O2 and normal oxygen concentration, respectively, for 6 h. The culture mediums were collected and centrifuged as the HCM and normoxia conditioned medium (NCM). The neurospheres of NSCs were obtained from the rat cerebral cortex by suspending culture. Immunohistochemical staining was used after adherence culture for 48 h to identify neurons and astrocytes in the progeny of NSCs. LY294002, a PI3-K inhibitor, and SP600125, a JNK inhibitor, were added into the HCM to culture NSCs for 48 h. The results showed that NSCs in the cerebral cortex could differentiate into β-Tubulin III immunoreactive neurons and GFAP immunoreactive astrocytes in three conditioned mediums, and the neurons proportion in progeny of NSCs was higher than astrocytes in all three groups. The proportion of neurons in 4% HCM was higher than that in NCM (P < 0.01). But the proportion of neurons in 1% HCM was less than that in NCM (P < 0.01). Both LY294002 and SP600125 inhibited NSCs to differentiate into high proportion neurons induced by 4% HCM (P < 0.01), but the inhibitory effect of LY294002 was stronger than that of SP600125 (P < 0.01). In conclusion, 4% HCM can induce NSCs to differentiate into more neurons mainly through the PI3-K pathway.