Hematopoietic stem cells (HSC) maintain a balance between self-renewal and lineage commitment, and the homeostatic mechanisms of this regulation are not well understood. Glycogen synthase kinase-3 (GSK-3) negatively regulates multiple signaling pathways in hematopoiesis, including Wnt signaling and cytokines such as Thrombopoietin (Tpo) and Erythropoietin, which signal through JAK2 to activate STAT5, Akt, and MAP kinase pathways. We have found GSK-3 plays a pivotal role in controlling the balance between HSC self-renewal and differentiation. Inhibition of Gsk3 in bone marrow transiently expands phenotypic HSCs and hematopoietic progenitor cells (HPCs) in a ss-catenin dependent manner, consistent with a role for Wnt signaling. However, in long-term assays, Gsk3 inhibition progressively depletes HSCs through activation of mTOR. Long-term HSC depletion is prevented by mTOR inhibition and exacerbated by ss-catenin knockout. Thus Gsk3 regulates both Wnt and mTOR signaling in HSCs, with opposing effects on HSC self-renewal, and we find that combined inhibition of mTOR and Gsk3 results in sustained HSC expansion in vivo. Furthermore, by combining GSK-3 and mTOR inhibitors, we are able to culture functional HSCs from mice or humans in cytokine- free medium and then reconstitute long-term, multilineage hematopoiesis in transplant recipients, providing a system for the ex vivo study of hematopoiesis and a potential method to expand human HSCs for clinical applications. The overall goal of this project is to examine the respective contributions of Wnt, mTOR, and Tpo/Mpl/JAK2 signaling in the response to GSK-3 inhibition and to define the role of GSK-3 in regulating these pathways within the hematopoietic system. Specific aim 1 will rigorously test both the independent and overlapping functions of Gsk3a and Gsk3b in bone marrow cells using conditional gene knockouts. Aim 2 will explore the role of Wnt and mTOR pathways downstream of GSK-3 in HSCs, and will identify the cell population that responds to GSK-3 and mTOR inhibitors under ex vivo conditions. Aim 3 will investigate the upstream pathways that regulate GSK-3 and mTOR, including cytokine signaling through JAK2 and Akt-dependent pathways, and will specifically distinguish the respective contributions of Wnt and Akt signaling through GSK-3. These studies will define the function(s) of GSK-3 in normal hematopoiesis and may lead to the development of novel therapeutic approaches to expand HSCs in hematopoietic disorders.