Antigen-presenting cells (APC) can express surface ligands with both T cell activating and inhibitory capacities, prompting the question of how responding T cells integrate opposing trans signals concurrently delivered by APC. To address this question in a quantitative fashion, we turned to protein transfer as a unique experimental approach that is well-suited for addressing such questions from a quantitative standpoint. Costimulatory (either B7-1*Fc(gamma1) or Fc(gamma1)*4-1BBL) and pro-apoptotic (Fc(gamma1)*FasL) Fc fusion proteins were quantitatively "painted" in varying ratios onto surrogate APC pre-coated with palmitated-protein A, the latter serving as a surface anchor. Evaluating the signaling potential of these various painted cells in a standard in vitro T cell proliferation assay, we demonstrated that at a given level of TCR triggering, the quantitative balance between costimulator (B7-1 or 4-1BBL) and FasL dictates the magnitude of the proliferative T cell response. Furthermore, when the costimulator density is kept constant, there is also a quantitative balance between TCR-directed and FasL signals. Interesting species-specific nai;ve versus memory T cell subset differences emerged with regard to susceptibility to Fas-mediated apoptosis and costimulator:FasL opposition. Taken together, these data demonstrate for the first time a quantitative interplay between activating and pro-apoptotic trans signals that dictates the magnitude of T cell responses.