BCR-ABL expression led to a dramatic up-regulation of the IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor receptor beta common (IL-3Rbetac) and IL-3 receptor beta (IL-3Rbeta) chains in murine embryonic stem cell-derived hematopoietic cells coincident with an expansion of multipotent progenitors and myeloid elements. This up-regulation required BCR-ABL tyrosine kinase activity and led to IL-3Rbetac/beta chain tyrosine phosphorylation in the absence of detectable IL-3 production. These results suggested that cytokine-independent IL-3 receptor activation could be a dominant signaling component in BCR-ABL-induced leukemogenesis. To unambiguously define the significance of IL-3 receptor-dependent signaling in BCR-ABL-induced leukemogenesis, BCR-ABL-transduced bone marrow cells deficient in either IL-3Rbetac chain or both IL-3Rbetac/beta chain expression were examined for their ability in generating myeloproliferative disease (MPD). These BCR-ABL-expressing knockout cells were capable of generating MPD similar to control cells, demonstrating that IL-3 receptor activation is not essential for BCR-ABL-induced MPD. However, the IL-3Rbetac/beta chain could act as a cofactor in BCR-ABL-induced leukemogenesis by activation of its many known oncogenic signaling pathways.