To investigate the effects of signal transducer and activator of transcription 3 (Stat3) on neural stem cell fate, stem cells were inoculated with an adenovirus vector expressing dominant negative form of Stat3 (Stat3F). One day later, a promoter assay revealed significant reduction of the transcriptional level in the transfected cells. Three days later, Western blot analysis and immunocytochemical analysis revealed that the protein level of microtubule-associated protein (MAP)2 and the number of MAP2-positive cells were increased significantly in the transfected cells whereas the protein level of glial fibrillary acidic protein (GFAP) and the number of GFAP-positive cells were decreased significantly. In addition, mRNA levels of Notch family members (Notch1, 2, and 3) and of inhibitory basic helix-loop-helix (bHLH) factors (Hes5, Id2, and Id3) were significantly downregulated at 3 days after viral inoculation with Stat3F; however, mRNA levels of bHLH determination factors (Math1 and Neurogenin3) and bHLH differentiation factors (NeuroD1 and NeuroD2) were significantly upregulated. These data indicated that suppression of Stat3 directly induced neurogenesis and inhibited astrogliogenesis in neural stem cells.