The analysis of human B cell populations of the blood relies on the expression of surface markers, mainly CD19, CD24, CD38, and CD27. According to these surface markers, three main B cell subsets can be identified in the blood: immature transitional B cells (CD19(+)CD24(high)CD38(high)), naïve B cells (CD19(+)CD24(int)CD38(int)) that have not encountered an antigen, and memory B cells (CD19(+)CD27(+)). To date, human B cells with regulatory functions have been essentially described within the CD24(high)CD38(high) transitional B cell subset. CD24(high)CD38(high) transitional B cells are able to produce interleukin 10 (IL-10) and to regulate in vitro Th1 and Th17 CD4(+) T cell activation. Here, we provide the methods to analyze and purify the CD24(high)CD38(high) transitional B cell subset for further in vitro experiments. We also provide a reliable method to detect B cell IL-10 production using intracellular cytokine staining.