Broadly neutralizing anti-HIV antibodies are rare and have proved hard to elicit with any immunogen. We have tested in vitro the notion that such antibodies or other antiviral proteins could be made by lentivirus-mediated gene transfer into human hematopoietic stem/progenitor cells (HSPCs), followed by differentiation of the transduced cells into B cells, the most potent antibody-producing cells. To do this, we have developed a highly efficient system for in vitro maturation of secreting B lymphocytes and plasma cells from CD34⁺ HSPCs. It is a 3-stage, in vitro culture system that supports normal human B-lineage development from HSPCs to antibody-secreting plasmablasts (∼36%) and plasma cells (∼20%). By transducing human cord blood CD34⁺ cells with lentiviral vectors encoding a secretory monoclonal anti-HIV antibody, b12 (IgG₁), we were able to program human B cells to produce in vitro up to 1.5 μg/mL of this broadly neutralizing antibody. Our results suggest that an HIV vaccine might be delivered by autologous transplantation of in vitro–programmed HSPCs, which would develop into antibody-secreting B cells in vivo and provide a continuous supply of anti-HIV neutralizing antibodies.