Increasing evidence shows the presence and significance of CD8+ T regulatory cells (CD8+ Tregs) in both human and rodent transplant recipients, as well as in autoimmune disease models. We, hereafter, review all available data on the phenotypic and functional characterization of CD8+ Tregs, and we also provide detailed protocols to purify them and analyze their suppressive function. Different subsets of dendritic cells (DCs) and CD4+ effector T cells may modulate the suppression mediated by CD8+ Tregs. By analyzing the proliferation of CFSE-labeled naïve CD4+CD25- T cells in coculture MLR and transwell experiments, we explored the mutual modulation of CD8+ Tregs, DC subsets, and CD4+ T effector cells. The suppressive function of CD8+ Tregs was mediated by both cell-contact-dependent and -independent mechanisms.