Background: In mice, a tri-layered (superficial, intermediate, and deep) vascular structure is formed in the retina during the third postnatal week. Shortterm treatment of newborn mice with vascular endothelial growth factor (VEGF) receptor inhibitors delays the formation of superficial vascular plexus and this allows us to investigate the developmental process of superficial and deep vascular plexuses at the same time. Using this model, we examined the effect of pharmacological depletion of retinal neurons on the formation of superficial and deep vascular plexuses. Results: Neuronal cell loss induced by an intravitreal injection of N-methyl-Daspartic acid on postnatal day (P) 8 delayed vascular development in the deep layer but not in the superficial layer in mice treated with KRN633, a VEGF receptor inhibitor, on P0 and P1. In KRN633-treated mice, neuronal cell loss decreased the number of vertical sprouts originating from the superficial plexus without affecting the number of angiogenic sprouts growing in front. Neuronal cell loss did not impair networks of fibronectin and astrocytes in the superficial layer. Conclusions: Our results suggest that inner retinal neurons play a crucial role in forming the deep vascular plexus by directing the sprouts from the superficial blood vessels to the deep layer. K E Y W O R D S astrocyte, hypoxia, neuronal cell, vascular development, vascular endothelial growth factor 1 | INTRODUCTION In the mouse retina, three planar (superficial, intermediate, and deep) vascular networks are established during the third postnatal week. 1,2 Superficial vascular plexus forms during the first postnatal week, and the vascularization in the deep layer starts after the vasculature covers the entire retinal surface (7-10 days of postnatal age). Afterward, the intermediate vascular plexus is formed. 2 Vascular endothelial growth factor (VEGF) plays an important role in the formation of retinal vascular network. Hypoxia and hypoxia-inducible factors (particularly their subunits HIF-1α and HIF-2α) regulate VEGF expression. 3-5 In the retina, neuronal cells and glial cells produce VEGF and contribute to the development of the retinal vasculature. 6 For example, previous studies have demonstrated that endothelial cells failed to migrate to the retina from the optic disc in retinal ganglion cell