Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-α (TNFa)-induced apoptosis. BimL, a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial apoptotic pathway.However, the molecular mechanism of BimL activating Bax activation during TNFα-induced apoptosis is not fully understood. In this study, the roleof BimL inBax activationduring TNFα- induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria after TNFα treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria. Co-immunoprecipitation results show that, the interaction between Bcl-xL and Bax decreased after TNFa treatment, while that between Bcl-xL and BimL increased. Bax did not co-immunoprecipitate with BimL beforeor after the TNF atreatment. Inaddition, the increased interaction between BimL and Bcl-xL was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Mostimportantly, there was noevidence of BimL redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that BimL displaces Bcl-xL in the mitochondria and promotes Bax translocation during TNFa-induced apoptosis.