Visual information is relayed from eyes to the brain by axons of the retinal ganglion cells (RGCs). The progenitor cells in neuroblast layer (NBL) of the embryonic retina produce RGCs, which migrate to the ganglion cell layer (GCL) prior to the neurite differentiation in mice. By in situ hybridization, we observed that Fezf2, a key gene for the development of projection neurons in cerebral cortex, was transiently expressed in NBL at E16.5. In contrast, Tbr1, a Fezf2 repressor, expressed in the GCL where no obvious Fezf2 signal was detected. Ectopic expression of Fezf2 in E13.5 retina decreased the expression of Hes5, a gene critical for the maintenance of retinal progenitor pool, as determined by real-time PCR assays of the transfected cells. Fezf2-overexpressing cells remained in NBL, instead of moving to GCL, suggesting that Fezf2 down regulation was required for RGC migration. Furthermore, the transfected cells colocalized with the neuronal marker Gap43 but not with the RGC makers such as Islet1 and Math5. Although axons of the transfected cells form the optic nerve, they did not reach superior colliculus at E17.5 when the normal RGCs had established retinotectal projections. We hypothesized that a transient expression of Fezf2 may induce the formation of neuronal cells by inhibition of Hes5, and the later down-regulation is required for generation and maturation of RGCs. This study may improve our understanding of the RGC development, which will help gene and cell therapy for retina degeneration diseases in future.