B-cell development is initiated in the fetal liver and neo-natal bone marrow and continues in the peripheral lymphoid organs upon exposure to antigens. The various stages of the development of B cells are associated with the several cell-surface markers that are regulated throughout the process. As with all differentiation processes associated with a large number of markers, multi-colour flow cytometry is an ideal technique for gaining insights into B-cell development. This data sheet describes a 12-colour flow cytometry panel for the analysis of the most prominent B-cell subsets in human peripheral blood and bone marrow. The panel included markers expressed uniquely in cell subsets like plasma cells (CD138) as well as co-expressed markers like CD24 and CD38. The analysis was done on a BD FACSCelesta™ analyzer using six BD Horizon™ Brilliant violet, two BD Horizon™ Brilliant ultraviolet dyes and four additional dyes that allowed the best resolutions of the B-cell populations. In samples derived from different donors, the panel identified different B-cell subsets. Visualisation and analysis of bias-free cell clusters, generated using t-distributed stochastic neighbourhood embedding algorithms (t-SNE) on the FlowJo™ software, enabled the comparison of cell distribution between different tissues. In addition to detecting the major B-cell populations, combining BD FACSCelesta™ with BD Horizon™ Brilliant high-performance dyes also enabled the identification of rare cell types like plasma cells.