SUMMARY The thrombopoietin receptor, c-Mpl, like other members of the cytokine receptor superfamily, requires the association and activation of Janus kinases for normal signal transduction. The membrane-proximal portion of the signaling domain, containing conserved box1 and box2 motifs, is sufficient to support the proliferation of cytokine-dependent cell lines and basal megakaryocytopoiesis in vivo. We hypothesized that activation of the Jak2 kinase alone might be sufficient for proliferative signaling. To test this premise we constructed chimeric receptors, in which the extracellular and transmembrane portions of Mpl were fused to the pseudokinase and kinase domains of murine Jak2 kinase. When expressed in the interleukin-3 (IL-3)-dependent cell line Ba/F3, the chimeric receptors were appropriately expressed on the cell surface and were able to initiate tyrosine kinase activity upon exposure to thrombopoietin (TPO). However, chimeric receptors lacking an intact box2 domain of Mpl were unable to support proliferation at any concentration of TPO. Only chimeric receptors containing both Jak2 kinase activity and the box2 region initiated proliferative signaling. Within the box2 motif, we determined that amino acids Glu56-Ile57-Leu58 of the Mpl cytoplasmic domain are critical for proliferation of the chimeric receptors. Furthermore, TPO-dependent induction of c-myc transcription is also dependent on this motif. These results indicate that Jak2 activation alone is not sufficient for TPO-induced proliferation and that one or more essential signaling pathways must arise from the cytoplasmic domain of Mpl that includes box2. Although the nature of the signal transduction pathway is not yet known, this second proliferative event is likely to regulate c-myc expression.