Objective To investigate whether erythropoietin (EPO) activated astrocytes could release some bioactive factors which affect differentiation of neural stem cells and protect these differentiated cells after hydroxyl free radical induced injury. Methods Primarily cultured astrocytes were prepared and purified, and the astrocytes conditioned medium was collected and used to culture neural stem cells. After 5 days in vitro the morphological changes of the neural stem cells were observed under a phase contrast microscope, NF-200 immunocytochemical staining was conducted to evaluate the differentiation of neural stem cells. In the mean time, FeSO4 and H2O2 were added to neural stem cells for 20 min, and these cells were cultured for 48 hours with or without EACM, then the viability of cells was determined with MTT assays, and survived cells were counted as well. Results The expression of NF-200 in the neural stem cells showed strong positive whereas control group exhibited less morphological changes and NF-200 immunocytochemical staining was almost negative in corresponding period. As for EACM protective effects on differentiated neural stem cells after hydroxyl freeinduced injuries generated by adding FeSO4 and H2O2, the absorbance value of the experimental group was 0.381±0.027 while the data was only 0.27±0.013 in control group (EM>P/EM><0.01). Conclusion These results suggest that EPO activated astrocytes can secrete some factors to either promote differentiation of neural stem cells into neuron or protect differentiated neu