Stem cell self-renewal and differentiation must be carefully controlled during development and tissue homeostasis. In the Drosophila optic lobe, neuroepithelial cells first divide symmetrically to expand the stem cell population and then transform into asymmetrically dividing neuroblasts, which generate medulla neurons. The mechanisms underlying this cell fate transition are not well understood. Here, we show a crucial role of some cell cycle regulators in this transition. We find that loss of function in replication protein A (RPA), which consists of three highly conserved protein subunits and functions in DNA replication, leads to disintegration of the optic lobe neuroepithelium and premature differentiation of neuroepithelial cells into medulla neuroblasts. Clonal analyses of RPA loss-of-function alleles indicate that RPA is required to prevent neuroepithelial cells from differentiating into medulla neuroblasts. Inactivation of the core cell cycle regulators, including the G1/S regulators E2F1, Cyclin E, Cdk2, and PCNA, and the G2/M regulators Cyclin A, Cyclin B, and Cdk1, mimic RPA loss-of-function phenotypes, suggesting that cell cycle progression is required for both maintaining neuroepithelial cell identity and suppressing neuroblast formation. We further find that RPA or E2F1 inactivation in the neuroepithelial cells correlates with downregulation of Notch signaling activity, which appears to result from Numb mislocalization. Thus, we have shown that the transition from neuroepithelial cells to neuroblasts is directly regulated by cell cycle regulators and propose a model in which the inhibition of neuroepithelial cell cycle progression downregulates Notch signaling activity through Numb, which leads to the onset of neurogenesis.