Type 1 regulatory T (Tr1) cells are adaptive regulatory T cells that are induced in the periphery upon chronic exposure to antigen (Ag) in a tolerogenic environment containing interleukin (IL)-10. Tr1 cells are Ag-specific; they produce high levels of IL-10 and TGF-β in the absence of IL-4 and suppress T-cell responses via a cytokine-dependent mechanism. During the last decade, several protocols have been developed to generate Tr1 cell lines in vitro. In this chapter, we outline protocols to generate non-Ag- and Ag-specific Tr1 cell lines and assays used to characterize Tr1 cell phenotype and functions.