Cell death facilitates the removal of infected cells; it can be triggered by many stimuli, which activate numerous biochemical pathways that lead to apoptotic (immunologically silent), as well as non-apoptotic (including pyroptosis or Caspase-1-dependent, and necroptosis or RIPK3/MLKL-dependent) forms of death (1) (Figure 1). Recently we revealed a new role for pyroptosis in hematopoietic stem and progenitor cells (HSPC) during hematopoietic stress induced by viral infection and chemotherapy causing cytopenia, immunosuppression and bone marrow failure (2). We now present preliminary data, currently in press at Cell, that RIPK3-dependent necroptotic cell death limits the self-renewal capacity of LT-HSC in lethally-irradiated recipient mice (Figure 2). This result unveils non-apoptotic cell death as a key biological process restricting HSC self-renewal, and has major clinical implications for improving the engraftment potential of HSC in transplantation settings using bone marrow, G-CSF-mobilized peripheral blood stem cells, cord blood units, and for gene therapy clinical trials. These data will change our view of one of the cornerstones of HSC biology: the self-renewal of HSC and how necroptosis impacts lifelong hematopoiesis and immunity. Our extensive preliminary data also establishes RIPK1 as a negative regulator of RIPK3/MLKL-dependent necroptosis. To access necroptotic cell death pathways, most previous studies utilize caspase inhibitors to block Caspase-8-mediated inhibition of RIPK3. Here we can now investigate necroptosis without treating primary cells or mice with caspase inhibitors, heralding a new approach to understanding necroptosis and the possible interaction of this non-apoptotic cell death pathway with executioner caspases such as caspase-3. This application seeks to use these necroptotic Ripk1-/- chimeric mice to specifically investigate the physiological consequences of necroptosis and its negative regulation by RIPK1 in hematopoieisis. We will investigate the following specific aims to understand how necroptosis affects HSPC: Aim 1. Define the role of RIPK1 and MLKL in the self-renewal of HSC and recovery from chemotherapy. Aim 2. Biochemical, morphological, and kinetic analysis of necroptosis ex vivo. Aim 3. Study the role of RIPK1 and MLKL in viral-infected HSPC.