ABSTRACT Stem cell proliferation is controlled through cell cycle arrest and activation. In the central nervous system of Drosophila melanogaster, neuroblast quiescence and activation takes place in defined spatial and temporal patterns. Two genes have been identified that regulate the pattern of neuroblast quiescence and proliferation. ana, which has been previously described by Ebens and coworkers (Ebens, A., Garren, H., Cheyette, B. N. R. and Zipursky, S. L. (1993). Cell 74, 15–28), encodes a secreted glial glycoprotein that inhibits premature neuroblast proliferation. We previously showed that trolsd causes a dramatic drop in the number of dividing cells in the larval brain late in development. This study presents evidence that this decrease results from a failure to activate proliferation in the quiescent neuroblast population at the appropriate time. However, trolsd does not affect the maintenance of cell division in already dividing mushroom body neuroblasts. The quiescent optic lobe and thoracic neuroblasts affected by trolsd proliferate in a trol mutant background if they have been activated by a lack of the ana proliferation repressor, demonstrating that trolsd does not affect cellular viability, nor does trol represent a celltype-specific mitotic factor. This also shows that trol acts downstream of ana to activate proliferation of quiescent neuroblasts in an anadependent pathway, possibly by inactivating or bypassing the ana repressor. These results suggest that trol and ana are components of a novel developmental pathway for the control of cell cycle activation in quiescent neuroblasts.