LIM-homeodomain (HD) and POU-HD transcription factors play crucial roles in neurogenesis. However, it remains largely unknown how they cooperate in this process and what downstream target genes they regulate. Here, we show that ISL1, a LIM-HD protein, is co-expressed with BRN3B, a POU-HD factor, in nascent post-mitotic retinal ganglion cells (RGCs). Similar to the Brn3b-null retinas, retina-specific deletion of Isl1 results in the apoptosis of a majority of RGCs and in RGC axon guidance defects. The Isl1 and Brn3b double null mice display more severe retinal abnormalities with a near complete loss of RGCs, indicating the synergistic functions of these two factors. Furthermore, we show that both Isl1 and Brn3b function downstream of Math5 to regulate the expression of a common set of RGC-specific genes. Whole-retina chromatin immunoprecipitation and in vitro transactivation assays reveal that ISL1 and BRN3B concurrently bind to and synergistically regulate the expression of a common set of RGC-specific genes. Thus, our results uncover a novel regulatory mechanism of BRN3B and ISL1 in RGC differentiation.