The overarching goal of this proposal is to evaluate a novel method to mobilize and procure a hematopoietic stem cell (HSC) allograft which we hypothesize will lead to rapid hematopoietic engraftment but cause less graft versus host disease (GVHD) compared to G-CSF mobilized peripheral blood stem cells (G-PBSC) following allogeneic HSC transplantation (HSCT). We plan to study the combination of the CXCR4 antagonist plerixafor and Flt3 ligand (CDX-301). This combination has not previously been studied but based on characteristics of both agents, we hypothesize the combination will safely mobilize a more favorable balance of HSC, conventional T-cells, regulatory T-cells (Treg), and natural killer (NK) cells which will optimize favorable graft versus leukemia (GVL) effects while mitigating deleterious GVHD reactions. A series of studies proposed in preclinical murine and non-human primate models will test our hypotheses. Despite curative potential, HSCT is associated with serious complications such as GVHD and disease relapse which compromise its success. Recently, G-PBSC has replaced bone marrow (BM) as the most commonly used donor graft source, but this trend may have been premature as emerging data suggest G-PBSC is associated with higher rates of chronic GVHD, and in pediatric patients and those with aplastic anemia, worse survival. The higher rates of GVHD observed following transplantation of G-PBSC may be due to the higher quantity of T-cells or other less well characterized factors compared to BM. In this proposal, we hypothesize that the combination of plerixafor and CDX-301 will lead to more robust hematopoietic stem/progenitor and NK-cell cell mobilization compared to plerixafor, G-CSF, or CDX-301 alone and will promote rapid hematopoietic reconstitution following transplantation with reduced risk of GVHD compared with G-PBSC. We will test these hypotheses using three specific aims. In Specific Aim 1, we will study the efficacy of the combination of CDX-301 and plerixafor in mobilization and expansion of hematopoietic stem cells and mature immune cell subsets in the peripheral blood of mice. In Specific Aim 2, we will study the potential of donor hematopoietic cells mobilized by the combination of CDX-301 and plerixafor to reduce GVHD and relapse in mismatched murine leukemia transplantation models. In Specific Aim 3: we will compare the mobilization of hematopoietic cells by plerixafor combined with either G-CSF or Flt3L in a clinically relevant non-human primate model. The successful completion of these aims will establish scientific and technical merits and will pave the way for Phase II of the Small Business Technology Transfer (STTR) process.