Overexpression of antiapoptotic proteins including Bcl-XL and/or Bcl-2 contributes to tumor initiation, progression, and resistance to therapy by direct interactions with proapoptotic BH3 proteins. Release of BH3 proteins from antiapoptotic proteins kills some cancer cells and sensitizes others to chemotherapy. Binding of Bcl-XL and Bcl-2 to the BH3 proteins Bad, Bid, and the three major isoforms of Bim was measured for fluorescent protein fusions in live cells using fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer. In cells the binding of the proteins at mitochondria is similar to the results from in vitro measurements. However, mutations in the BH3 region of Bim known to inhibit binding to Bcl-XL and Bcl-2 in vitro had much less effect in MCF-7 cells. Moreover, the BH3 mimetic ABT-737 inhibited Bad and Bid but not Bim binding to Bcl-XL and Bcl-2. Thus, the selectivity of ABT-737 also differs markedly from predictions made from in vitro measurements.