We have investigated the ability of glycoprotein (GP) Ibα, a megakaryocytic gene product, to sequester the signal transduction protein 14-3-3ξ and to influence megakaryocytopoiesis. Using a Gp1ba^–/– mouse colony, we compared the rescued phenotypes produced by a wild-type human GP Ibα allele or a similar allele containing a 6-residue cytoplasmic tail truncation that abrogates binding to 14-3-3ξ. The observed phenotypes illustrate an involvement for GP Ibα in thrombopoietin-mediated events of megakaryocyte proliferation, polyploidization, and the expression of apoptotic markers in maturing megakaryocytes. We developed a hypothesis for the involvement of a GP Ibα/14-3-3ξ/PI-3 kinase complex in regulating thrombopoietin-mediated responses. An observed increase in thrombopoietin-mediated Akt phosphorylation in the truncated variant supported the hypothesis and led to the development of a model in which the GP Ibα cytoplasmic tail sequestered signaling proteins during megakaryocytopoiesis and, as such, became a critical regulator in the temporal sequence of events that led to normal megakaryocyte maturation.