The retina is an exquisitely patterned tissue, with neuronal somata positioned at regular intervals to completely sample the visual field. Cholinergic amacrine cells are spectacular exemplars of precision, distributing in two radial layers and tangentially, forming regular mosaics. Here, we investigated how the intracellular phosphatase Pten and the cell adhesion molecule Dscam cooperate to regulate amacrine cell patterning. Using double mutants to test epistasis, we found that Pten and Dscam function in parallel pathways to regulate amacrine cell positioning. Mechanistically, Pten regulates endocytic remodeling of cell adhesion molecules (Dscam, Megf10, Fat3), which are aberrantly redistributed in Pten conditional-knock-out (cKO) cholinergic amacrine cells. Furthermore, extracellular vesicle content, including that of cell adhesion and signaling molecules, is altered in Pten cKO retinas. Consequently, Wnt signalling is attenuated, the pharmacological disruption of which phenocopies Pten cKO amacrine cell patterning defects. Pten thus controls endocytic trafficking of critical cell adhesion/signaling molecules to control amacrine cell spacing.