The culture of isolated photoreceptors in vitro has remained elusive in neuroscience. By using defined hyaluronic acid (HA) hydrogels, photoreceptor survival and maturation in vitro is dramatically increased, as evidenced by upregulation of outer segment markers at the RNA and protein levels. While substrate stiffness is known to be a key factor influencing cell survival in vitro, it is shown that isolated photoreceptors do not respond to modifications in hydrogel stiffness modifications but depend, instead, on HA for survival. While the molecular pathways that are induced by HA on photoreceptors are unknown, mTOR activation is identified as the molecular mechanism underlying the pro-survival effect, and it is demonstrated that the canonical Wnt and RhoA pathways are intermediaries. This work establishes a valuable method for isolated photoreceptor culture in vitro, which will be useful in translational and basic retinal research. The pathways identified herein may be useful targets in retinal degeneration.