It has been shown that the glutathione S-transferase π (GSTπ) interacts with and suppresses the activity of c-Jun NH2-terminal kinase (JNK). GSTπ deficient mice (GSTπ-/-) have higher levels of circulating white blood cells than wild type. A peptidomimetic inhibitor of GSTπ disrupts the interaction between GSTπ and JNK and mimics in wild type mice the increased myeloproliferation observed in GSTπ-/- animals. Until now, the molecular basis of this effect has not been defined. In an in vitro hematopoiesis assay, interleukin-3, granulocyte-colony stimulating factor and granulocyte- macrophage-colony stimulating factor were more effective at stimulating proliferation of hematopoietic cells in GSTπ-/- mice than in wild type. The GSTπ inhibitor increased myeloproliferation induced by these cytokines in wild type but not in GSTπ-/- animals, suggesting a key role for GSTπ in the effect of this drug. The JNK inhibitor, SP600125 which itself caused little inhibition of cytokine-induced myeloproliferation, prevented all myelostimulant effects of the GSTπ inhibitor. A more sustained phosphorylation of the STAT family of proteins was also observed in GSTπ-/- bone marrow derived mast cells exposed to IL-3. This was associated with an increased proliferation and a down-regulation of expression of negative regulators of the JAK-STAT pathway. The increased activation of JNK and STATs in GSTπ-deficient mice provides a viable mechanism for the increased myeloproliferation in these animals. These data also confirm the important role that GSTπ plays in the regulation of cell signaling pathways in bone marrow.