Background and Objective: Currently, all live FRET experiments using fluorescent proteins have utilized for exploring two-protein complex. Here, we report the development of a novel FRET technology for studying complexes among three proteins. The system utilizes CFP, YFP and mRFP (monomer red fluorescent protein). CFP->YFP FRET1, YFP->mRFP FRET2 and CFP->YFP->mRFP chained FRET signals are monitored separately by flow cytometry. The technology has been validated by constructing plasmids encoding several FRET-positive and -negative controls, including CFP-YFP-mRFP, CFP-T2TD-YFP-mRFP and CFP-YFP-T2TD-mRFP, whereas T2TD (TRAF2 TRAF domain) acts as FRET insulator because of its structure with a distance of 9 nm from the head to the end. Furthermore, this technology has been used to examine the trimer formation of TRAF2 in living cells. Results: 1. The validity of CFP->YFP->mRFP 3-way FRET: We for the first time validate the practicality of using flow cytometry to determine CFP->YFP->mRFP chained FRET by directly visualizing 2-step-FRET signals and the quenching of CFP->YFP FRET1 signals. 2. The determination of three protein complexes: with the system, we examined the trimer formation of tumor-necrosis-factor-associated factor 2 in living Hela cells. TRAF2 was tagged separately by CFP, YFP and mRFP. Co-transfection of all three plasmids displayed the 2-step-chained FRET signals, but not in cells co-transfected with any two of them and blank fluorescent protein. Based on these data, one publication has been published: He L, Wu X, Simone J, Hewgill D, Lipsky PE. Determination of tumor necrosis factor receptor-associated factor trimerization in living cells by CFP->YFP->mRFP FRET detected by flow cytometry. Nucleic Acids Res. 2005 Apr 1;33(6):e61. Conclusions: we have achieved method to determine interactions among three proteins in living cells using flow cytometry and CFP-YFP-mRFP FRET system.