Costimulation is a fundamental principle of T‐cell activation. In addition to T‐cell receptor engagement, the interaction between CD80 and/or CD86 with CD28 and/or cytotoxic T‐lymphocyte antigen 4 (CTLA‐4) receptors is required to regulate T‐cell activation and tolerance. While the importance of costimulation is clearly established, the exact molecular mechanism is unknown. We demonstrate that T‐cell proliferation and the ability of CD8+ T‐effector cells to kill were enhanced slightly by CD80 but dramatically by CD86 costimulation. To further analyse the cellular process of costimulation, we developed a single‐cell assay to analyse Ca2+ signals following costimulation with bi‐specific antibodies. We found that this stimulation method worked in every human T‐cell that was analysed, making it one of the most efficient T‐cell activation methods to date for primary human T cells. The enhanced proliferation and killing by costimulation was paralleled by an increase of Ca2+ influx following CD86 costimulation and it was dependent on CD28/CTLA‐4 expression. The enhanced Ca2+ influx following CD86 costimulation was abrogated by an antibody that interfered with CD28 function. The differences in Ca2+ influx between CD80 and CD86 costimulation were not dependent on the depletion of Ca2+ stores but were eliminated by the application of 10 μm 2‐aminoethyldiphenyl borate which has recently been shown to enhance stromal interaction molecule 2 (STIM2)‐dependent Ca2+ entry while reducing STIM1‐dependent Ca2+ entry. Our data indicate that differences in the efficiency of costimulation are linked to differences in Ca2+ entry.