PU.1 is a transcription factor that plays a critical role throughout hematopoiesis. PU.1 was initially described as the causative oncogene in murine erythroleukemias (28). PU.1 is absolutely required for the development of several myeloid lineages as well as B lymphocytes. In these lineages PU.1 regulates the expression of many genes required for terminally differentiated cells to function (reviewed in (9)). PU.1 also plays a role in the development of lineages such as T lymphocytes and red blood cells where PU.1 is not expressed in mature cells. In these lineages PU.1 is expressed in early progenitors and facilitates their differentiation (15, 40). Indeed, PU.1 even plays a pivotal role in the hematopoietic stem cell. It is required for the proper engraftment and maintenance of HSC in the bone marrow microenvironment (8). As a community we still have a very limited understanding of how HSC progress to become lineage committed progenitors that give rise to the distinct functional cells of the hematopoietic system. We know that developmental changes take place in the HSC and that a variety of multipotent progenitors are produced that lack the ability to self-renew. In addition, we have recently proven that adult HSC can robustly function as hemangioblasts (13) - that is make both blood and blood vessels. We have also developed a new model for hematopoietic stem cell (LSC or oval cell) based liver regeneration in the mouse. In this system PU.1-/- HSC produce hepatocytes better than WT HSC. This may be due to the inability of PU.1-/- HSC to make blood, thereby being encouraged to make liver. To control and follow PU.1 function we have knocked-in a Tamoxifen inducible PU.1 fusion protein (PUERT) and both gfp and LacZ to the PU.1 locus. These models and tools will allow us to further dissect the role of PU.1 in stem cell fate and differentiation. In this proposal we will address the following Specific Aims: Aim 1. Is PU.1 linked to the initial development of the definitive and transplantable HSC? Aim 2. Define the role of PU.1 in the transition from fetal HSC to adult HSC phenotype? Is this transition reversible? Is PU.1 required to establish marrow hematopoiesis? Aim 3. Does PU.1 influence the developmental fate of adult stem cells?