Most cell death in vertebrates proceeds through the intrinsic pathway of apoptosis and results from unregulated increase of mitochondrial membrane permeability. Bcl2-associated X protein (Bax) and Bcl2-antagonist/killer protein (Bak), the effector proapoptotic members of the Bcl-2 family, are, in their active state, the principal accomplices for this permeabilization process. How exactly Bax and Bak are activated has been a matter of major investigation in the last decade, and suitable tools offered by quantitative cytometric methodologies have significantly contributed to the understanding of the function of Bcl-2 family members. Here, we review the most relevant findings in this field and highlight one common trait that has emerged from the diverse new theories: a crucial role in the control of Bax/Bak activation has to be attributed to the BH3-only subset of the Bcl-2 family. BH3-only proteins exert their proapoptotic activity by hierarchical and tightly tuned interactions with other Bcl-2 family members and operate as sensors of intracellular/extracellular death signals and vectors of information to the core apoptotic machinery. Given their essential role in apoptosis, BH3-only molecules are proposed as molecular targets for the cure of diseases associated with abnormal cell death, as in the case with neurodegenerative conditions. As well, they are explored as possible tools for cancer therapy, according to the concept that molecules mimicking the BH3 domain of these proteins could selectively and efficiently cooperate in the cell killing by chemotherapeutic drugs. A few BH3 mimetics are currently being tested in clinical trials of hematologic and solid tumors. Nevertheless, the knowledge about the cellular and molecular mechanisms that regulate responsiveness to BH3 therapy has to be further expanded and will benefit from recent advances in cytometric quantitative technologies.