Primary human B cells are an attractive target for gene-therapeutic applications, but have been found to be relatively resistant toward transduction with lentiviral vectors (LVVs), even though a number of different envelope pseudotypes were tested. Moreover, low transgene expression in primary human B cells has impeded the use of LVVs for this target cell. We investigated the transduction potential of gibbon-ape leukemia virus (GALV) Env-pseudotyped LVVs for primary human B cells. By establishing optimized transduction kinetics and multiplicities of infection, we were able to regularly obtain transduction efficiencies of more than 50% in CD40L-activated B cells. Noteworthy, with the use of GALV-pseudotyped LVVs we could achieve a more than 10-fold higher yield of transduced activated B cells in direct comparison with LVVs pseudotyped with measles virus glycoproteins. Phenotyping of transduced primary B cells revealed a majority of memory B cells, a long-lived phenotype, presumed to be well suited for enduring therapeutic interventions. Finally, by combining the enhancer (Eμ) and the matrix/scaffold-attachment regions (MARs) of the human immunoglobulin heavy chain with the promoter of spleen focus-forming virus (SFFV) we aimed at generating a novel LVV particularly suitable for B cell transgenesis. We show that the optimized vector facilitated significantly higher transgene expression in various B cell lines and, more importantly, primary human B cells (mean factor of three). In summary, we have established a novel protocol for the efficient lentiviral transduction of primary human B cells and have improved transgene expression in B cells by a specific vector modification.