Introduction. Cell lines and cultures are indispensable tools in cell physiology research. Cultures of B lymphocytes are essential for the study of B-cell response, antibody generation, as well as for the creation of therapeutic monoclonal antibodies. At the same time, the activation of B-lymphocytes and their long-term cultivation in vitro continues to be an unsolved problem. The aim of the study – development of a protocol for the stimulation of human B lymphocytes in vitro, characterization of the phenotype and functional characteristics of stimulated B cells for the subsequent use of the protocol for determining the number of memory B cells, sequencing of Ig genes, as well as obtaining immortalized clones of B lymphocytes. Material and methods. Cells stably transfected with the CD40L gene were generated by lentiviral transduction. B lymphocytes were isolated from human peripheral blood by centrifugation in a density gradient of ficoll-verografin with further enrichment by negative selection using magnetic beads. B lymphocytes were stimulated in vitro with feeder cells carrying surface CD40L in the presence of exogenous recombinant interleukin (IL) -21. The number and phenotype of stimulated B lymphocytes were determined using multicolor flow cytometry. The secretion of IgM and IgG in cultures of B lymphocytes was assessed using enzyme-linked immunosorbent assay. Results. Using lentiviral transduction, HEK293, K562 and A549 cells stably transfected with the CD40L gene were obtained. Based on the expression of the CD40L molecule, as well as growth properties, the transfected A549 cell line was selected for further research as a feeder. When feeder cells and B lymphocytes were co-cultured in the presence of exogenous IL-21, stimulation of B lymphocytes was observed. B lymphocytes began to proliferate and after 7 days their number increased by an average of 8 times. The stimulated B lymphocytes changed their morphology. They acquired an irregular shape with pseudopodia, grouped around feeder cells, and began to move actively. Upon IL-21/CD40L stimulation, B lymphocytes changed their surface phenotype and by day 10, about 40 % of B cells had differentiated into plasmablasts (CD19 CD27 CD38 ). At the same time, the proportion of CD20 cells decreased to 20 %. The percentage of cells with surface Ig expression also decreased. In contrast, the secretion of IgM and IgG increased during stimulation. By the 12 day, the proliferative potential of B cells was exhausted, and they died. Сonclusion. In vitro cultivation of B lymphocytes in the presence of exogenous IL-21, as well as feeder cells expressing the CD40L molecule, is a convenient system for obtaining activated B lymphocytes that can be used in different applications. + + + + th