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Abstract Chimeric antigen receptor (CAR) cell therapy represents a significant advancement in immunotherapy against hematologic malignancies and autoimmune diseases. Nevertheless, single-target CAR therapy encounters several challenges, including antigen escape and disease antigen heterogeneity, resulting in drug resistance and disease recurrence. Therefore, broadening the recognition spectrum of CAR cells has driven the development of dual- or multiple-target CAR cell therapy as an important research direction to enhance the clinical outcome for patients. B-cell maturation antigen (BCMA) is highly expressed in late-stage B-cell malignancies, and plasma disorders, making it an ideal target for developing immunotherapies against multiple myeloma (MM) and B-cell-related plasma cell disorders. Preclinical and early clinical results have shown that combination approaches targeting CD20 and CD79b break resistance and outperform single-target therapies in aggressive B-cell lymphomas. Therefore, we developed a human pluripotent stem cell (PSC)-derived NK cell platform to engineer triple-target CARs (BCMA, CD20 and CD79b) to overcome treatment resistance and improve the anti-tumor activity, aiming to create a standardized, “off-the-shelf” allogeneic cell therapy to treat B-cell related malignancies and disorders. Using CRISPR/Cas12b, we engineered PSCs by simultaneously integrating BCMA-CAR and CD20/CD79b-CAR (loop) into validated genomic safe harbor loci, knocking out B2M and CIITA genes, and overexpressing HLA-E to achieve MHC-I/II silencing while maintaining immune-evasive properties, and introducing constitutive IL15RF to promote sustained in vivo persistence. We found that these modified PSCs failed to sustain the generation of hematopoietic cells and the subsequent lymphoid progenitors, resulting in limited production of PSC-derived NK cells with poor viability and effector function. Further investigation revealed that BCMA protein starts to express during PSC-HSC differentiation, peaks at the early HSC-NK progenitor stage, and gradually disappears during NK cell maturation and expansion. Therefore, we hypothesized that the interaction between endogenously expressed BCMA and ectopically expressed BCMA-CAR during early PSC-NK differentiation may interfere with the differentiation process, ultimately compromising NK cell functionality. To address this challenge, we developed a novel small molecule-regulated gene switch to specifically induce the expression of BCMA-CAR at the final PSC-derived NK expansion stage. This approach enabled the generation of regulated BCMA-CAR-expressing, trispecific BCMA/CD79b/CD20-targeting PSC-derived CAR-NK cells (AXA-NK02a). Comprehensive in vitro evaluations demonstrated that AXA-NK02a exhibits robust cytotoxicity. In nonspecific cytotoxicity assays using K562 cells, AXA-NK02a displayed superior natural killer cell activity. Compared to single CAR-expressing T cells, AXA-NK02a mediated faster killing of SUDHL-CD79b (CD79b⁺), SUDHL-CD20 (CD20⁺), U266 (BCMA⁺), and Jeko-1 (CD79b⁺CD20⁺BCMA⁺) tumor cells. In vivo, AXA-NK02a achieved rapid and profound Jeko-1 tumor growth inhibition and demonstrated robust antitumor activity. In summary, the AXA-NK02a, featuring inducible control technology, combines rapid action, multi-antigen coverage and sustained activity, supporting its development as a promising “off-the-shelf” PSC-derived cell therapy for B cell-related malignancies and autoimmune diseases.