Achieving homeostasis within neural stem cell (NSC) lineages is essential for nervous system development and maintenance. It requires an exquisite balance between NSC self-renewal and differentiation. The molecular and cellular mechanisms underlying the control of NSC homeostasis remain poorly understood. Elucidation of these mechanisms will provide novel insights into the development and maintenance of the nervous system as well as offer the keys to molecularly targeted therapy for diseases resulting from NSC homeostasis failure, including brain tumors and neurodevelopmental, psychiatric, and neurodegenerative disorders. We propose to elucidate the basic mechanisms underlying the genetic control of NSC homeostasis, using Drosophila larval brain type II neuroblasts (NBs) as a model. Drosophila NBs have been instrumental in discovering signaling molecules such as Numb and Notch, and cellular mechanisms such as asymmetric cell division, that are centrally involved in NSC homeostasis. Like mammalian NSCs, fly type II NBs generate transit-amplifying intermediate progenitors (IPs), which help to generate a vast number of differentiated progenies. Notch signaling is critical for maintaining the stemness of type II NBs. Inhibition of Notch signaling results in NB fate not being properly maintained, whereas aberrant Notch activation causes ectopic NB formation and brain tumorigenesis. The function of Notch in regulating NSC homeostasis appears to be conserved in mammals. However, there is much to be learned about the mechanisms of action of Notch and its in vivo relationship with Numb, which remains enigmatic and controversial. We have found that canonical Notch signaling is necessary but not sufficient for Notch-directed NSC regulation and that a novel non-canonical Notch signaling pathway is also involved. Our main hypothesis is that non-canonical Notch signaling acts coordinately with canonical Notch signaling to mediate distinct aspects of NSC homeostasis control, and that Numb regulates both of these two pathways. Many fundamental questions are raised: What cellular programs do the canonical and the non-canonical pathways regulate? What are the key molecular targets of these pathways? Can we recapitulate the effect of Notch by manipulating these key targets? What roles does Numb play in these two pathways? Three specific aims will help address these questions. Aim 1 will genetically and biochemically elucidate a non-canonical Notch signaling pathway that regulate NSC homeostasis. Aim 2 will test the hypothesis that the non-canonical Notch pathway and the canonical pathway act coordinately to maintain NSC homeostasis. Aim 3 will test the hypothesis that Numb acts in a novel protein complex to regulate key downstream mediators in the canonical and non-canonical Notch pathways. Upon successful completion of these Aims, we will generate new mechanistic insights into the control of NSC homeostasis by Notch and Numb. We anticipate that this will open up entirely new directions for studying the fundamental roles of Numb and Notch in NSC biology and cancer biology.