B cells are generated every day in the bone marrow, but only a small fraction integrates the peripheral B-cell pool. In the murine spleen, we can find several B-cell subsets representing various maturation stages and/or cell functions. The spleen is a complex lymphoid organ organized in two main structures with different functions: the red and white pulp. The red pulp is flowed with blood while the white pulp is organized in primary follicles, with a B-cell area composed of follicular B cells and a T-cell area surrounding a periarterial lymphatic sheath. The frontier between the red and white pulp is defined as the marginal zone and contains the marginal zone B cells. Because B cells, localized in different areas, are characterized by distinct expression levels of B-cell receptor (BCR) and other surface markers, splenic B-cell subsets can be easily identified and purified by flow cytometry analyses and cell sorting (FACS). Here, we will focus on marginal zone B cells and their precursors giving some experimental hints to identify, generate, and isolate these cells. We will combine the use of FACS analysis and confocal microscopy to visualize marginal zone B cells in cell suspension and tissue sections, respectively.