Six3, a homeodomain-containing transcriptional regulator belonging to the Six/so family, shows a defined spatiotemporal expression pattern in the developing murine telencephalon, suggesting that it may control the development of specific subsets of neural progenitors. We find that retrovirus-mediated misexpression of Six3 causes clonal expansion of isolated cortical progenitor cells by shortening their cell cycle and by prolonging their amplification period, while maintaining them in an immature precursor state. Our results show that the observed effects exerted by Six3 overexpression in mammalian brain depend strictly on the integrity of its DNA-binding domain, suggesting that Six3 action likely relies exclusively on its transcriptional activity. In vivo upregulation of Six3 expression in single progenitor cells of the embryonic telencephalon keeps them in an undifferentiated state. Our observations point to a role of Six3 in the control of the subtle equilibrium between proliferation and differentiation of defined precursor populations during mammalian neurogenesis.