FoxO is a family of forkhead transcription factors that negatively regulate proliferation and survival signals in hematopoietic cells. We and others have previously shown that inhibition of the three members of this family (FoxO1, FoxO3 and FoxO4) by leukemogenic tyrosine kinase fusion genes results in enhanced proliferative and survival signaling in leukemic cells. For example, the transforming activities of the lymphoma associated NPM-ALK (nucleophosmin-anaplastic lymphoma kinase) fusion, BCR-ABL, or FLT3-ITD, are mediated in part by inactivation of FoxO through phosphorylation and ubiquitin mediated degradation by constitutively active Akt (Gu TL, et al. Blood 2004), with subsequent induction of proliferative and survival signals. Furthermore, inhibition of FoxO is required for efficient transformation of hematopoietic cells. However, the roles of FoxO in adult hematopoiesis are unknown. We have initiated studies to examine the role of FoxO in the context of normal hematopoiesis and leukemogenesis using a triple conditional knockout mouse for each of the FoxO1, FoxO3 and FoxO4 alleles. The FoxO alleles are flanked by lox-P sites and conditional excision is mediated by Cre expression under the control of the interferon inducible Mx1 promoter. Based on the normal function of FoxO family members to repress proliferative and survival signals, we hypothesized that the deletion of FoxO subfamily members would lead to an enhanced proliferation and survival in the hematopoietic compartment, and might contribute to the development of a myeloproliferative and/or lymphoproliferative phenotype in vivo. Triple homozygous conditional FoxO knockout mice were generated in an Mx1-Cre background to allow for excision of the FoxO alleles in the hematopoietic stem cell compartment after treatment with pIpC. Complete excision of each of the three alleles in the hematopoietic compartment was confirmed. However, in contrast with our working hypothesis, we observed that loss of function of FoxO family members was associated with a relatively subtle hematopoietic phenotype with 12 months of follow-up. The phenotype includes a non-fatal mild myeloproliferative phenotype that is progressive over time and characterized by modest splenomegaly, extramedullary hematopoiesis and increased mature myeloid populations in bone marrow and spleen. In addition, there are subtle alterations in both B and T lymphoid cell populations, including a decrease in both immature and mature B cells in the spleen and bone marrow; and abnormalities of CD4+CD8+ double positive and CD4+ and CD8+ T cells in the thymus. Examination of stem and progenitor populations also revealed subtle differences in the HSC and CLP progenitor populations at 4 weeks post pIpC. Thus, these data indicate that complete loss of FoxO function in the adult hematopoietic compartment results in a relatively subtle hematopoietic phenotype. They further demonstrate that although inhibition of FoxO family members is required for efficient transformation of hematopoietic cells by leukemogenic fusion tyrosine kinases, loss of FoxO function alone is not sufficient to induce a leukemic phenotype.