Regulatory B cells that produce IL-10 are now recognized as an important component of the immune system. We have identified a rare antigen-specific regulatory B-cell subset with a unique CD1d^hiCD5⁺CD19^hi phenotype in the spleens of wild-type mice. We call these cells B10 cells because they are responsible for most B cell IL-10 production, they appear to only produce IL-10 after 5 h of in vitro stimulation, and to distinguish them from other potential regulatory B cell subsets. B10 progenitor (B10pro) cells have also been identified within the spleen CD1d^hiCD5⁺CD19^hi B-cell subset, and within other lymphoid tissues. Herein, four methods for identifying and isolating regulatory IL-10-producing B10 cells in mice are provided. The first two methods are used to identify and enumerate B10 and B10pro cells based on their cell surface phenotypes and cytoplasmic IL-10 staining. The last two methods are used to isolate viable B10 cells for adoptive transfer and functional studies. These methods should facilitate the study of B10 cells in inflammation, autoimmune disease, immune responses, and cancer therapy.