Neurotrophic factors can promote the proliferation and differentiation of neural stem cells (NSCs). Here we report that the possibility of using bFGF in combination with BDNF and NGF to promote proliferation and differentiation of NSCs in vitro. C57BL/6 mouse NSCs were cultured, passaged and stained by immunofluorescence for nestin and GFP. According to different neurotrophic factors added to NSCs, seven experiment groups (NGF, BDNF, bFGF, bFGF+NGF, bFGF+BDNF, NGF+BDNF and NGF+BDNF+bFGF) and a blank control group were established. One week after induction and differentiation, results showed that there was significant difference in the percentage of NSCs differentiating into neurons among the experiment groups. The percentage in the multi-factor groups was significantly higher than that in the single-factor groups (p<0.05), among which the percentage was the highest in NGF+BDNF+bFGF group. In the two-factor groups, the percentage in bFGF+NGF and bFGF+BDNF groups was significantly higher than that in NGF+BDNF group (p<0.05). The NSCs growth curves showed that cells proliferated continuously with the time of culture prolonging, but there was significant difference between the group containing bFGF and that without bFGF. Our results demonstrate that combined use of NGF/BDNF/bFGF significantly improved the ability of NSCs proliferation and differentiation.