Protein tyrosine phosphorylation plays a crucial role in signaling from the receptor for erythropoietin (Epo), although the Epo receptor (EpoR) lacks the tyrosine kinase domain. We have previously shown that the Jak2 tyrosine kinase couples with the EpoR to transduce a growth signal. In the present study, we demonstrate that Lyn, a Src family tyrosine kinase, physically associates with the EpoR in Epo-dependent hematopoietic cell lines, 32D/EpoR-Wt and F36E. Coexpression experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of the EpoR and that both LynA and LynB, alternatively spliced forms of Lyn, bind with the membrane-proximal 91-amino acid region of the EpoR cytoplasmic domain. In vitro binding studies using GST-Lyn fusion proteins further showed that the Src homology (SH)-2 domain of Lyn specifically binds with the tyrosine-phosphorylated EpoR in lysate from Epo-stimulated cells, whereas the tyrosine kinase domain of Lyn binds with the unphosphorylated EpoR. Far-Western blotting and synthetic phosphopeptide competition assays further indicated that the Lyn SH2 domain directly binds to the tyrosine-phosphorylated EpoR, most likely through its interaction with phosphorylated Y-464 or Y-479 in the carboxy-terminal region of the EpoR. In vitro binding studies also demonstrated that the Lyn SH2 domain directly binds to tyrosine-phosphorylated Jak2. In vitro reconstitution experiments in COS7 cells further showed that Lyn induces tyrosine phosphorylation of Stat5, mainly on Y-694, and activates the DNA-binding and transcription-activating abilities of Stat5. In agreement with this, Lyn enhanced the Stat5-dependent transcriptional activation when overexpressed in 32D/EpoR-Wt cells. In addition, Lyn was demonstrated to phosphorylate the EpoR and Stat5 on tyrosines in vitro. These results suggest that Lyn may play a role in activation of the Jak2/Stat5 and other signaling pathways by the EpoR.