The myeloproliferative disorder of mice lacking the Src homology 2 (SH2)-containing 5′ phosphoinositol phosphatase, SHIP, underscores the need for closely regulating phosphatidylinositol 3-kinase (PI3K) pathway activity, and hence levels of phosphatidylinositol species during hematopoiesis. The role of the 3′ phosphoinositol phosphatase Pten in this process is less clear, as its absence leads to embryonic lethality. Despite Pten heterozygosity being associated with a lymphoproliferative disorder, we found no evidence of a hematopoietic defect in Pten^+/- mice. Since SHIP shares the same substrate (PIP₃) with Pten, we hypothesized that the former might compensate for Pten haploinsufficiency in the marrow. Thus, we examined the effect of Pten heterozygosity in SHIP^-/- mice, predicting that further dysregulation of PIP₃ metabolism would exacerbate the pheno-type of the latter. Indeed, compared with SHIP^-/- mice, Pten^+/-SHIP^-/- animals developed a myelodysplastic phenotype characterized by increased hepatosplenomegaly, extramedullary hematopoiesis, anemia, and thrombocytopenia. Consistent with a marrow defect, clonogenic assays demonstrated reductions in committed myeloid and megakaryocytic progenitors in these animals. Providing further evidence of a Pten^+/-SHIP^-/- progenitor abnormality, reconstitution of irradiated mice with marrows from these mice led to a marked defect in short-term repopulation of peripheral blood by donor cells. These studies suggest that the regulation of the levels and/or ratios of PI3K-derived phosphoinositol species by these 2 phosphatases is critical to normal hematopoiesis. (Blood. 2004;103:4503-4510)