Nicotinamide mononucleotide adenylyltransferase (Nmnat) is a class of enzymes with three members (Nmnat1-3). Nmnat1 is in nucleus and associated with Leber congenital amaurosis, a form of early-onset retinal degeneration, while Nmnat2 is in cytoplasm and a well-characterized neuroprotective factor. The differences in their biological roles in the retina are unclear. We performed short hairpin RNA (shRNA)-based loss-of-function analysis of Nmnat2 during mouse retinal development in retinal explant cultures prepared from early (E14.5), middle (E17.5), or late (postnatal day [P]0.5) developmental stages. Nmnat2 has important roles in the survival of retinal cells in the early and middle stages of retinal development. Retinal cell death caused by Nmnat2 knockdown could be partially rescued by supplementation with NAD or nicotinamide mononucleotide (NMN). Survival of retinal cells in the late stage of retinal development was unaffected by Nmnat2, but differentiation of Müller glia was controlled by Nmnat2. RNA-Seq analyses showed perturbation of gene expression patterns by shRNAs specific for Nmnat1 or Nmnat2, but gene ontology analysis did not provide a rational explanation for the phenotype. This study showed that Nmnat2 has multiple developmental stage-dependent roles during mouse retinal development, which were clearly different from those of Nmnat1, suggesting specific roles for Nmnat1 and Nmnat2.