We recently found that the Rb protein is important for the regulation of retinal progenitor cell proliferation and rod photoreceptor development in the mouse retina. These two functions are separate for Rb and in this study we further characterize the role of Rb in retinal development. At postnatal day 12 in the retinae of Chx10-Cre;RbLox/- mice, immature cells are found in the outer nuclear layer where rods normally are differentiating. This results in alternating patches of the outer nuclear layer (ONL) that are lacking rod inputs. At this stage of development, horizontal cell processes at the outer plexiform layer do not mature appropriately and they extend into the outer nuclear layer. These disruptions in horizontal cell differentiation can persist for several weeks into the adult stage. While there are several secondary effects of the loss of Rb on retinal development including, limited cell death in the ONL, Müller glial cell activation, persistence of immature cells in the ONL, and altered nuclear morphology of cells in the ONL, we suggest that the defect in horizontal cell synapse formation at the OPL results from fewer rod inputs. Mice with other developmental defects in photoreceptor cell fate specification or glial cell activation do not exhibit a similar alteration in horizontal cell differentiation. Therefore, the retinae from Chx10-Cre;RbLox/- mice represent a unique model to study the role of rod photoreceptor inputs in horizontal cell differentiation and synapse formation.