In the mouse retina, at least ten distinct types of bipolar interneurons are involved in the transmission of visual signals from photoreceptors to ganglion cells. How bipolar interneuron diversity is generated during retinal development is poorly understood. Here, we show that Irx5, a member of the Iroquois homeobox gene family, is expressed in developing bipolar cells starting at postnatal day 5 and is localized to a subset of cone bipolar cells in the mature mouse retina. In Irx5-deficient mice, defects were observed in the expression of some, but not all, immunohistological markers that define mature Type 2 and Type 3 OFF cone bipolar cells, indicating a role for Irx5 in bipolar cell differentiation. The differentiation of these two bipolar cell types has previously been shown to require the homeodomain-CVC transcription factor, Vsx1. However, the defects observed in Irx5-deficient retinas do not coincide with a reduction of Vsx1 expression, and conversely, the expression of Irx5 in cone bipolar cells does not require the presence of a functional Vsx1 allele. These results indicate that there are at least two distinct genetic pathways (Irx5-dependent and Vsx1-dependent) regulating the development of Type 2 and Type 3 cone bipolar cells.