Pbx1 is a proto-oncogene that was originally discovered at the site of chromosomal translocations in pediatric acute leukemias. It codes for a homeodomain protein that binds DNA as a higher-order complex with a large subset of Hox proteins to modulate their DNA binding affinities and specificities. This project employs loss-of-function mouse models to investigate the contributions of Pbx1 and the related Pbx2 and Pbx3 genes to hematopoiesis and growth control. Ongoing studies show that Pbx1 is an essential gene whose absence results in embryonic lethality during late gestation due to severe anemia. Insufficient production of mature erythrocytes in Pbx1-/ embryos is the result of cell-autonomous defects in myeloerythroid progenitors. The most severely affected are common myeloid progenitors (CMP), which display reduced proliferation and decreased frequency in Pbx1-/- mice. The studies proposed in Specific Aim #1 investigate the role of Pbx1 in lymphopoiesis to determine whether similar defects are present in Pbx1/- common lymphoid progenitors (CLP) or their progeny. A more general role for Pbx1 in growth control is suggested by multiple organ hypoplasias in Pbx1/-embryos and the premature entry of Pbx1-/- MEFs into replicative senescence in vitro, an INK4a/ARF-mediated process. The studies in Specific Aim #2 will address the possibility that Pbx1 functions in part through modulation of INK4a/ARF expression to regulate growth potential in vitro and in vivo. Pbx2 nullizygous mice are viable and fertile but their bone marrow stem/progenitor cells display enhanced self-renewal in vitro. The studies in Specific Aim #3 will investigate the possible role for Pbx2 in restricting growth of stem cells, its implications for ex vivo stem cell expansion, and the consequences for adult bone marrow hematopoiesis. Pbx3, along with Pbx1, is expressed in fetal liver hematopoietic progenitors and Pbx3 nullizygous mice display neonatal lethality whose underlying pathogenesis has not yet been determined. The studies in Specific Aim #4 will investigate the role of Pbx3 in fetal liver hematopoiesis through analysis of Pbx3-/- mice and compound Pbx1/Pbx3 intercrossed mice. Together, the proposed studies will address the hypothesis that Pbx proteins regulate the timing and/or extent of progenitor cell proliferation versus differentiation and serve as the basis for understanding the role of mutant Pbx1 proteins in human leukemogenesis.