In mice, all of the six retinal neuron types are generated from common multipotent retinal progenitors, and their differentiation from progenitors is regulated by both extrinsic and intrinsic factors. Previously, we showed that targeted deletion of the atonal (ato) homologue math5 blocked the differentiation of most retinal ganglion cells (RGCs), revealing an essential role for math5 in RGC differentiation. In this study, we used the Cre-loxP recombination system to trace the fate of math5-expressing cells in retina. Our results demonstrated that math5 expression was associated with the differentiation of multiple retinal neuron types, including RGCs, photoreceptor, horizontal, and amacrine cells, implying that math5 expression alone is not sufficient to determine the RGC fate. Math5 expression was restricted to postmitotic cells in developing retina, suggesting that cell fate commitment of retinal neurons occurs after the terminal mitosis. The insufficiency of and requirement for math5 in RGC differentiation indicates that, like ato in the development of Drosophila R8 photoreceptors, math5 plays a role in determining the RGC competence state of retinal progenitors and that additional positive and negative factors are required in determining RGC fate. Furthermore, we show that loss of Math5 function severely reduced the RGC expression of the transcription factors Brn-3b, Gfi-1, Isl-1, Isl-2, Nscl-1, Nscl-2, and RPF-1, suggesting that Math5 expression is required to activate a comprehensive transcription network of RGC differentiation.