Although much is known about the transcriptional regulation that coordinates retinal cell fate determination, very little is known about the developmental processes that establish the characteristic laminar architecture of the retina, in particular, the specification of neuronal positioning. The LIM class homeodomain transcription factor Lim1 (Lhx1) is expressed in postmitotic, differentiating, and mature retinal horizontal cells. We show that conditional ablation of Lim1 results in the ectopic localization of horizontal cells to inner aspects of the inner nuclear layer, among the retinal amacrine cells. The ectopic cells maintain a molecular phenotype consistent with horizontal cell identity; however, these neurons adopt a unique morphology more reminiscent of amacrine cells, including a dendritic arbor positioned within the inner plexiform layer. All other retinal cell populations appear unaltered. Our data suggest a model whereby Lim1 lies downstream of horizontal cell fate determination factors and functions cell autonomously to instruct differentiating horizontal cells to the appropriate laminar position in the developing retina. This study is the first to describe a cell type-specific genetic program that is essential for targeting a discrete retinal neuron population to the proper lamina.