Objective To explore the possibility of motor neuron differentiation from neonatal mouse telencephalon neural stem cells and the novel inducing factors. Methods Neural stem cells were isolated from neonatal mouse telencephalon using floating culture methods. Three differentiation groups were divided according to different inducing factors. Group 1 containing growth medium+5% fetal bovine serum(FBS) served as control. Group 2 containing growth medium+5% FBS+ retinoid acid(RA)+Shh+dbcAMP was inducing factor group. Medium collected from skeletal muscle cultures was used for motor neuron induction in Group 3. Double immunofluoesence staining of microtubule associated protein 2(MAP2) and homeo box(HB9) were used to detect the differentiation of motor neuron. Twelve samples were selected randomly for cell counting. Results Motor neurons with MAP2 and HB9 coexpression were found in differentiation cultures. The ratio of motor neuron differentiation was 1% in Group 1, 4.7% in Group 2 and 2.9% in Group 3. The difference between Group 2 or Group 3 and Group 1 was of statistic significance. Conclusion Neonatal mouse telencephalon neural stem cells can differentiate into motor neurons. Skeletal muscle cells might secret motor neuron inducing factors.