Macrophages (Mp) are professional antigen presenting cells, but when they accumulate at sites of inflammation, they can inhibit T cell proliferation. In experimental autoimmune uveoretinitis (EAU), this limits the expansion of T cells within the target organ. To define requirements for the elaboration of this outcome, we have generated populations of Mp in vitro that could also regulate T cell responses; stimulating CD4+ T cell activation and cytokine production, but simultaneously suppressing T cell proliferation. When T cells are removed from the influence of such cells, normal T cell responses are restored. We show that TNFR1 signalling is a critical checkpoint in the development of such Mp, as TNFR1-/- Mp are unable to suppress T cell proliferation. This deficit in antigen presenting cell results in a lack of production of prostaglandin E2 (PGE2), and nitric oxide, which are critical effector mechanisms that inhibit T cell division. However, TNFR1 signalling is not required for the inhibitory function of Mp, since we could circumvent the requirement for this receptor, by maturing Mp in the presence of exogenous IFN-gamma and PGE2. This produced TNFR1-/- Mp that inhibited T cell proliferation and indicates that TNFR1 delivers a signal that is necessary for the development but not the execution of this function.