Wnt signaling plays an essential role in the development of mammalian central nervous system. We investigated the impact of activation/inhibition of the Wnt signaling pathway on neuronal/glial differentiation in neurospheres derived from neonatal mouse forebrains. For short term alterations, neurospheres were stimulated with recombinant Wnt-3a, Wnt-5a and the Wnt inhibitor Dickkopf-1 (Dkk1). Furthermore, neurospheres were transduced with retroviral vectors encoding Wnt-3a, Wnt-7a and their inhibitors Dkk1 and soluble Frizzled related protein-5 (sFRP5). Long-term activation of Wnt pathway by Wnt-7a or by treatment with GSK3 inhibitors promoted a moderate increase of the neuronal differentiation and blocked gliogenesis. In contrast, Wnt pathway inhibition in neurospheres, induced by retroviral overexpression of either Dkk1 or sFRP5, robustly increased the gliogenesis at the expense of neurogenesis. In summary, our data demonstrate that activation or inhibition of Wnt/beta-catenin signaling in neurospheres regulates neuronal and glial differentiation, respectively. Thus, our results suggest that Wnt signaling may also contribute to regulate these processes in the neonatal brain.