Erythropoietin (Epo) modulates calcium influx through the calcium-permeable channel TRPC2. Here, the mechanisms through which Epo activates TRPC2 were examined. Erythroblasts were isolated from the spleens of phenylhydrazine-treated mice, and Epo stimulation resulted in a significant and dose-dependent increase in intracellular calcium ([Ca]_i). This increase in [Ca]_i was inhibited by pretreatment with the phospholipase C (PLC) inhibitor U73122 but not by the inactive analog U73343, demonstrating the requirement for PLC activity in Epo-modulated calcium influx in primary erythroid cells. To determine whether PLC is involved in the activation of TRPC2 by Epo, cell models were used to examine this interaction. Single CHO-S cells which express transfected Epo-R and TRPC2 were identified by detection of green fluorescent protein (GFP) and blue fluorescent protein (BFP), respectively. [Ca]_i was quantitated with Fura Red fluorescence. Epo-induced calcium influx through TRPC2 was inhibited by pretreatment with U73122, or by down regulation of PLC1 by RNAi. PLC activation results in the production of inositol 1,4,5-trisphosphate (IP₃), and TRPC2 has IP₃R binding sites. To determine whether IP₃R is involved in Epo-R signaling, TRPC2 mutants were prepared with partial (aa 1001-1036) or complete (> aa 1000) deletions of the C-terminal IP₃R binding domains. In cells expressing TRPC2 IP₃R binding mutants and Epo-R, no significant increase in [Ca]_i was observed following Epo stimulation. TRPC2 coassociated with Epo-R, PLC, and IP₃R, and the association between TRPC2 and IP₃R was disrupted in these mutants. Our data demonstrate that Epo-R modulates TRPC2 activation through PLC and that interaction of IP₃R with TRPC2 is required. They also demonstrate that Epo-R, TRPC2, PLC, and IP₃R interact to form a signaling complex.