Erythroid progenitor cell expansion depends upon co-signaling by Epo receptor (EpoR) and Kit, but underlying mechanisms are incompletely understood. To quantitatively analyze EpoR contributions to co-signaling, phosphotyrosine (Tyr(P)) mutants were expressed as human epidermal growth factor (hEGF) receptor-mEpoR EE chimeras at matched and physiological levels in FDCW2 hematopoietic progenitor cells and were assayed for proliferative activities in the absence or presence of endogenous Kit stimulation. Two Tyr(P)-null (but Jak2-coupled) EpoR forms each retained <or=25% of the wild-type activity, whereas the add-back of single Tyr(P) sites in the EpoR forms EE-T-Y343 (Stat5 binding site), EE-Y479 (p85/phosphatidylinositol 3-kinase binding site), or EE-Y464 (Src kinase binding site) significantly enhanced activities (to 100, 95, and 50% of EE-WT (wild type) levels, respectively). EE-Y343&Y401 and EEF343&F401 double add-back and deletion constructs were also prepared and were shown to possess 90 and <or=50% of wild-type activity. In contrast, efficient Kit co-signaling activity was retained only by EE-T-Y343 and EE-Y343&Y401 EpoR forms. EE-T-Y343 together with EE-T-Y343F and EE-WT EpoR forms were also analyzed in embryonic stem cell-derived erythroid G1E-2 cells with highly comparable outcomes, including the ability of EE-T-Y343 (but not EE-T-Y-343F) to synergize with Kit. Despite specific connection of EE-T-Y343 to Stat5, the contributions of Kit to EpoR-dependent proliferation did not involve Kit effects on Stat5 activation (but was limited by the mutation of Kit Tyr(P)-567 and Tyr(P)-569 Src kinase recruitment sites). Instead, co-signaling appears to depend upon the downstream integration of Kit signals with the targets of an EpoR/Jak2/Y343/Stat 5 response axis.