Thrombopoietin (TPO) is a recently characterized member of the hematopoietic growth factor family that serves as the primary regulator of megakaryocyte (MK) and platelet production. The hormone acts by binding to the Mpl receptor, the product of the cellular proto-oncogene c-mpl. Although many downstream signaling targets of TPO have been identified in cell lines, primary MKs, and platelets, the molecular mechanism(s) by which many of these molecules are activated remains uncertain. In this report we demonstrate that the TPO-induced activation of phosphoinositol 3-kinase (PI3K), a signaling intermediate vital for cellular survival and proliferation, occurs through its association with inducible signaling complexes in both BaF3 cells engineered to express Mpl (BaF3/Mpl) and in primary murine MKs. Although a direct association between PI3K and Mpl could not be demonstrated, we found that several proteins, including SHP2, Gab2, and IRS2, undergo phosphorylation and association in BaF3/Mpl cells in response to TPO stimulation, complexes that recruit and enhance the enzymatic activity of PI3K. To verify the physiological relevance of the complex, SHP2-Gab2 association was disrupted by overexpressing a dominant negative SHP2 construct. TPO-induced Akt phosphorylation was significantly decreased in transfected cells suggesting an important role of SHP2 in the complex to enhance PI3K activity. In primary murine MKs, TPO also induced phosphorylation of SHP2, its association with p85 and enhanced PI3K activity, but in contrast to the results in cell lines, neither Gab2 nor IRS2 are phosphorylated in MKs. Instead, a 100-kDa tyrosine-phosphorylated protein (pp100) co-immunoprecipitated with the regulatory subunit of PI3K. These findings support a model where PI3K activity is dependent on its recruitment into TPO-induced multiphosphoprotein complexes, implicate the existence of a scaffolding protein in primary MKs distinct from the known Gab and IRS proteins, and suggest that, in contrast to erythroid progenitor cells that employ Gab1 in PI3K signaling complexes, utilization of an alternate member of the Gab/IRS family could be responsible for specificity in TPO signaling.