Objective The aim of this study was to explore induction and differentiation of endogenous neural stem cells(ENSCs) in the hippocampus soluble factors(HSF) from the hippocampus of adult Wistar rats by mimicking an intracranial microenvironment. Methods After Wistar rats sacrificed, the hippocampus tissue was obtained in cold DMEM/F12. After centrigued and filtered, the HSF was stored at -20℃. The ENSCs was obtained from the hippocampus tissue of a neonate Wistar rat. Collected the tissue, digested and obtained the ENSCs. After we observed the morphology, the ENSCs were cultured in different concentration (0、50、100、200、400 μl/mL) of HSF for 6 days, and compared the expression of Nestin and CD133 by immunocytochemistry. Meanwhile,we compared the Nestin and CD133 protein by western blot. And then we explored the optimal concentration of HSF by the numbers of all groups on the second and sixth day. Furthermore, we did the differentiated experiment using the same concentration of HSF. Results The number of neurospheres in the 100 μg/mL group was significantly higher than those in the other groups on the 6th day. Immunofluorescence revealed that the neurospheres from ENSCs in the 100 μg/mL group more highly expressed nestin and CD133 than control. This result was confirmed by western blot analysis. The neurospheres can differentiate into glia-like cells in 100 μg/mL HSF and 1% FBS expressing GFAP, S100 and P75 NGFR by immunofluorescence. Conclusion These data indicated that HSF alone, mimicking a destination of ENSCs in vitro, could induce and differentiate neurospheres from ENSCs, as a new method to get NSCs and glia-like cells differentiated from ENCs to repair the diseases of center nervous system.