CD28-costimulatory signals interact with antigen-specific TCR signals to enhance T cell activation, proliferation and differentiation. The regulation of CD28-costimulation is controlled by CTLA-4 through its shared affinity for the CD28-ligands CD80 and CD86. CTLA-4-ig (abatacept) has emerged as an effective treatment for rheumatoid arthritis. The aims of this study were to consider the factors that influence CD28-costimulation requirements during in vitro T cell stimulation in order to identify strategies that may predict or improve clinical responses to abatacept-treatment. The efficacy of abatacept during in vitro T cell stimulation inversely correlated with parameters that increased the strength of TCR-stimulation. The simultaneous inhibition of TCR- and CD28-signals by Cyclosporine A and abatacept respectively promoted the inhibition of T cell activation above the level seen by either agent alone. The active form of vitamin D3, 1,25(OH)2D3, acted in a comparable manner to CsA to increase CD28-costimulation requirements by specifically inhibiting TCR-driven activation. These findings suggest that clinical responses to abatacept treatment may be determined by the strength of TCR stimulus that underlies T cell activation. Furthermore, that vitamin D3 may represent a useful adjunct to enhance clinical responses to abatacept.