In general, TGFβ is synthesized as a procytokine that requires proteolytic activation, release of the mature cytokine from its noncovalently associated latent-associated peptide, and binding to TGFβRII to mediate suppressive activity. We tracked this process in mice containing primed CD8 regulatory T cells (Tregs) by immunoblotting in primary whole cell lysates for pro-TGFβ, latent-associated peptide and mature TGFβ. Generation of CD8 Tregs promoted processing of the 50 kDa pro-TGFβ protein into a 12.5 kDa mature TGFβ species in vivo. Despite the inability to detect mature TGFβ in the sera of mice with primed CD8 Tregs and in the synthetic culture medium of stimulated CD8 Tregs, we demonstrated engagement of TGFβRII through immunoblotting for Smad2 phosphorylation. This process relied on continual TCR triggering, which also induced Smad3 phosphorylation. To understand the movement of mature TGFβ, we showed that in contrast to IFN-γ, mature TGFβ does not remain a soluble cytokine but is likely to be rapidly adsorbed by neighboring cells. These data show the exquisite local control directed toward TGFβ by the immune system and underscore the fine specificity involved in its detection.