Pax6 encodes a highly conserved transcriptional regulator that contains two DNA binding domains, the paired domain (PD) and homeodomain (HD). In mammals, Pax6 is widely expressed in a complex spatiotemporal pattern during the development of the eye, olfactory bulbs and central nervous system and plays important roles in pattern formation, cell fate determination and cell cycle progression in these regions. Here, we characterized a Pax6-dependent signaling pathway through which Pax6 controls progenitor cell proliferation in the developing telencephalon. Comparison of cell cycle parameters between Pax6+/+ and Pax6sey/sey progenitors suggested that correct levels of Pax6 are crucial in regulating progenitor cell proliferation. To address the possible molecular basis of the cell cycle defect observed in Pax6sey/sey embryos, the expression of a number of cell cycle genes was analyzed by qPCR in the lateral cortex of Pax6+/+ and Pax6sey/seyembryos, which confirmed the significantly altered expression levels of these genes. Of these, Cdk6 was further identified as a direct target of Pax6 and the interaction of putative binding sites with Pax6 protein was confirmed by EMSA in vitro and by qChIP in vivo. In addition, the functional role of these Pax6 binding sites, through which Pax6 represses the transcription of Cdk6, was further evaluated by luciferases assays. Activation of Cdk6 is required for pRb phosphorylation as well as induction of the pRB/E2F pathway, and in turn promotes the G1-S cell-cycle transition. An increase in pRb phosphorylation accompanied by changes in pRb subcellular distribution and up-regulation of E2F downstream targets were observed in the cortex of sey/sey embryos. In contrast, reduced Cdk6 expression and pRb phosphorylation was found in HEK293 cells overexpressing Pax6. Collectively, these findings provided new insight into the molecular mechanism of Pax6-dependent regulation of progenitor cell proliferation in the developing telencephalon.