Little is known regarding the modulation of regulatory T cell (Treg) activity. Maturing dendritic cells (DC) secrete a cocktail of cytokines (R-cocktail) that liberates T cells from Treg suppression. IL-6 is one component, but others remain unknown. We performed microarray analysis of maturing DC and identified IL-12p40, TNFα, and Ebi3 as candidates. We developed a DC-free in-vitro suppression assay to test their function. As expected, addition of supernatant from maturing DC allowed T cells to resist suppression. Use of IL-6 increased T cell resistance significantly. Its combination with IL-12 or TNFα increased T cell resistance further. IL-12 and TNFα were interchangeable, suggesting redundant activity. The R-cocktail exerted its effect on T cells since a short pre-exposure before co-culture released them from suppression. On the other hand, Treg pre-exposure increased their suppressive capacity, indicating that the effect exerted on T cells was dominant. Unexpectedly, when we tested Ebi3, in the form of IL-27, we observed sustained Treg suppressive activity, an effect that was exerted specifically on Treg, as indicated by the pre-exposure experiment. This observation helps clarify the reported anti-inflammatory capacity of IL-27. Thus, pro-inflammatory cytokines exert distinct effects on T cells and Treg, promoting counter-balancing activities used by the immune system to finely tune its reactivity.