Thrombospondin-4 (TSP-4), a large pentameric glycoprotein of the extracellular matrix, has been described as a neurite outgrowth-promoting molecule. However, the means by which TSP-4 promotes neurite outgrowth in the developing eye is unclear. Here we show that TSP-4 is present at the appropriate time in development and displays a localization pattern within the developing mouse retina consistent with a role in retinal ganglion cell (RGC) neurite outgrowth. Furthermore, results indicate that while TSP-4 alone does not support adhesion or neurite extension, it enhances the ability of laminins to promote adhesion and neurite outgrowth of embryonic retinal cells. The mechanism of enhancement is, in part, based on the ability of TSP-4 to enhance the three-dimensionality and/or clustering of laminins within the substrate matrix. These results support a model where TSP-4 acts as an organizer of adhesive and axon outgrowth-promoting molecules in the ECM to optimize retinal ganglion cell responses.