The progenitor cells in the cerebral cortex coordinate proliferation and mitotic exit to generate the correct number of neurons and glial cells during development. However, mechanisms for regulating the mitotic cycle of cortical progenitors are not fully understood. Otx1 is one of the homeobox-containing transcription factors frequently implicated in the development of the central nervous system. Mice bearing a targeted deletion of Otx1 exhibit brain hypoplasia and a decrease in the number of cortical neurons. We hypothesized that Otx1 might be crucial to the proliferation and differentiation of cortical progenitors. Otx1 knockdown by in utero electroporation in the mouse brain reduced the proportion of the G1 phase while increasing the S and M phases of progenitor cells. The knockdown diminished Tbr1+ neurons but increased GFAP+ astrocytes in the early postnatal cortex as revealed by lineage tracing study. Tbr2+ basal progenitors lacking Otx1 were held at the transit-amplifying stage. In contrast, overexpression of wildtype Otx1 but not an astrocytoma-related mutant Y320C inhibited proliferation of the progenitor cells in embryonic cortex. This study demonstrates that Otx1 is one of the key elements regulating cortical neurogenesis, and a loss-of-function in Otx1 may contribute to the overproduction of astrocytes in vivo.