Objective:To search for a method to induce human embryonic stem cells (hESCs) differentiating into neural stem cells (NSCs) in vitro. Methods:HESCs were induced to differentiate into NSCs by three-step differentiation under a condition simulating the microenvironment and different development stages of neural cells in vivo. The surface markers of hESCs and NSCs were detected by morphological observation, immunocytochemistry assay, flow cytometry and RT-PCR. The plasticity of NSCs was evaluated by differentiating test.Results:The hESCs retained expression of SSEA-4, TRA-1-81 proteins and Nanog genes after cultured for 50 passages. Flow cytometry revealed the positive rate of SSEA-4 was 83.44%.After induced by the three-step differentiation the purity of nestin-positive cells was higher than 90%. Flow cytometry revealed that the positive rate of nestin was 89.38%. The differentiated cells retained the characteristics of NSCs after repeated passaging and could be further induced into neurons, astrocytes and oligodendrocytes. Conclusion:The present three-step differentiation method can induce hESCs into high purity NSCs, while retaining the plasticity of stem cells.