Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Whilst it is clear that CTLA-4 is a critical regulator of T cell responses, the immunological contexts in which CTLA-4 controls immune responses are not well defined. Here we show that whilst CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression, in this context CTLA-4 blocking antibodies had no impact on the response. In contrast, in settings where CTLA-4+ cells were present as “regulators”, inhibition of resting T cell responses was dependent on CTLA-4 expression and specifically related to the number of antigen presenting cells. At low numbers of APC or low levels of ligand, CTLA-4-dependent suppression was highly effective whereas at higher APC numbers or high levels of ligand, inhibition was lost. Accordingly, the degree of suppression correlated with the level of CD86 expression remaining on the antigen presenting cells. These data reveal clear rules for the inhibitory function of CTLA-4 on Treg which are predicted by its ability to remove ligands from antigen presenting cells.