Human B lymphocytes are attractive targets for immunotherapies in autoantibody-mediated diseases. Gene editing technologies could provide a powerful tool to determine gene regulatory networks regulating B cell differentiation into plasma cells, and identify novel therapeutic targets for prevention and treatment of autoimmune disorders. Here, we describe a new approach that uses CRISPR-Cas9 technology to target genes in primary human B cells in vitro for identifying plasma cell regulators. We found that sgRNA and Cas9 components can be efficiently delivered into primary human B cells through RD114-pseudotyped retroviral vectors. Using this system, we achieved approximately 80% of gene knockout efficiency. We disrupted expression of a triad of transcription factors, IRF4, PRDM1, and XBP1, and showed that human B cell survival and plasma cell differentiation are severely impaired. Specifically, that IRF4, PRDM1, and XBP1 were expressed at different stages during plasma cell differentiation, IRF4, PRDM1, and XBP1-targeted B cells failed to progress to the pre-plasmablast, plasma cell state, and plasma cell survival, respectively. Our method opens a new avenue to study gene functions in primary human B cells and identify novel plasma cell regulators for therapeutic applications. Display omitted Here, we describe a new approach that uses CRISPR-Cas9 technology to target genes in primary human B cells in vitro for identifying plasma cell regulators. We disrupted expression of a triad of transcription factors, IRF4, PRDM1, and XBP1, and showed that human B cell survival and plasma cell differentiation are severely impaired.