Zebrafish can regenerate damaged photoreceptors following constant intense light treatment. During regeneration, the Müller glial cell dedifferentiates and reenters the cell cycle to produce neuronal progenitor cells, which continue to divide, migrate to the photoreceptor layer and differentiate into rod and cone photoreceptors. The mechanisms regulating dedifferentiation and proliferation of Müller glia, the formation and amplification of progenitor cells, and their subsequent differentiation to photoreceptors remain unknown. One signaling pathway associated with these processes during both development and regeneration is the Notch-Delta signaling pathway. While Notch has different functions in various cell types, it primarily regulates cell proliferation, cell fate choice and differentiation. To better understand the potential roles Notch signaling may play and differentiation. To better understand the potential roles Notch signaling may play during regeneration, γ-secretase inhibitors (GSIs), which block Notch signaling, were injected into retinas during light treatment and these retinas exhibited increased numbers of PCNA-positive INL cells during regeneration. The increased number of PCNA-positive INL cells was due to, at least in part, extra Müller glia reentering the cell cycle, suggesting Notch signaling may maintain Müller glial quiescence. At 14 and 28 days post injection, the number of Müller glia were significantly reduced in GSI-treated retinas relative to controls, while both UV and blue single cones were increased, suggesting that Notch signaling is required for Müller glia redifferentiation and plays a role in balancing cell fate choice of progenitor cells. Undamaged retinas injected intraperitoneally with GSIs exhibited significantly greater numbers of PCNA-positive INL cells, which were also Ascl1 and Stat3-positive. These two transcription factors are both required for the presence of these proliferative cells. At 7, 9, and 11 days after initial injection, there were very few Ath5-positive cells, an early neuronal cell marker, and several TUNEL-positive cells suggested that the proliferating cells may be undergoing cell death instead of committing to a neuronal lineage. However, the addition of TNFα significantly increased the number of PCNA-positive cells and Ath5-positive clusters relative to GSI injections alone. Overall, these data demonstrate that Notch signaling is important to Müller glial quiescence, cell fate choice and differentiation in the regenerating zebrafish retina.