Previous analysis of mutant mice has revealed that the bHLH genes Mash1 and Math3, and the homeobox gene Chx10 are essential for generation of bipolar cells, the interneurons present in the inner nuclear layer of the retina. Thus, a combination of the bHLH and homeobox genes should be important for bipolar cell genesis, but the exact functions of each gene remain largely unknown. We have found that in Mash1-Math3 double-mutant retina, which exhibits a complete loss of bipolar cells, Chx10 expression did not disappear but remained in Müller glial cells, suggesting that Chx10 expression per se is compatible with gliogenesis. In agreement with this, misexpression of Chx10 alone with retrovirus in the retinal explant cultures induced generation of the inner nuclear layer cells, including Müller glia, but few of them were mature bipolar cells. Misexpression of Mash1 or Math3 alone did not promote bipolar cell genesis either, but inhibited Müller gliogenesis. In contrast, misexpression of Mash1 or Math3 together with Chx10 increased the population of mature bipolar cells and decreased that of Müller glia. Thus, the homeobox gene provides the inner nuclear layer-specific identity while the bHLH genes regulate the neuronal versus glial fate determination, and these two classes of genes together specify the bipolar cell fate. Moreover, Mash1 and Math3 promoted the bipolar cell fate, but not the other inner nuclear layer-specific neuronal subtypes in the presence of Chx10, raising the possibility that the bHLH genes may be involved in neuronal subtype specification, in addition to simply making the neuronal versus glial fate choice.