Here, we establish that Rb suppresses RTC division and death by inhibiting E2f1, not E2f2 or E2f3. When these defects were rescued, most retinal neurons, including rods, survived, differentiated, and functioned normally. Thus, unexpectedly, retina-specific differentiation factors function independently of Rb. However, comprehensive assessment of the Rb/E2f1 double-null rescued retina revealed a differentiation defect in cholinergic starburst amacrine cells (SACs). Recent breakthroughs have revealed that these interneurons are critical for direction selectivity and developmentally important rhythmic bursts [34–36]. However, their differentiation is poorly understood. Contrary to the prevailing view that Rb promotes differentiation through E2f-independent tissue-specific transcription factors, we show that Rb facilitates SAC development through E2f3. Defects in Rb null SACs correlated with specific E2f3 expression in these cells, and E2f3 expression was absent in neurons that differentiated without Rb. E2f3 is also present in a specific subset of other CNS neurons, implying that this may be a general mechanism by which Rb facilitates neurogenesis. To define the mechanism in even more detail, we determined which E2f3 isoform Rb targets to control SAC differentiation. E2f3b mediates Rb function in quiescent fibroblasts [19], yet no prior studies to our knowledge have dissected E2f3a or E2f3b functions in vivo. Using an isoform-specific null mouse we show that Rb drives SAC differentiation through E2f3a. Thus, independent of E2f1-mediated effects on division and death, Rb does regulate neuronal differentiation, but only in specific neurons and, unexpectedly, through E2f3a, not tissue-specific differentiation factors.