This addition to UNIT 3.12 will describe the assays needed to evaluate CD4.CD25.T cell non-responsiveness and function. Unlike the conventional T cells described in , Basic Protocol 1, CD4.CD25. cells do not proliferate to TCR stimuli alone. The conditions required to induce proliferation are described. This addition will also describe the assay in which CD4.CD25.T cells are co-cultured with conventional T cells in order to assess their suppressive function. Finally, this addition will describe the culture conditions for the activation and expansion of CD4.CD25. cells.