Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation and differentiation through binding to and stimulation of the cell surface receptor c-MPL. Activating mutations in c-MPL constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-MPL that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study we focused on c-MPL tyrosine 591 (Y591). We found Y591 of wild-type c-MPL to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using an SH2/PTB domain binding microarray, we identified novel c-MPL binding partners for phosphorylated Y591, including Src homology phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through siRNA treatment of Ba/F3-MPL cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by SYK. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development.