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Abstract Background: We previously attempted to prove the existence of abnormal B cells, which are thought to be myeloma-initiating cells that originates from transformed mature B cells by reprograming. To elucidate the origin of myeloma cells, we induced a reciprocal chromosomal translocation t(11;14) in normal B lymphocyte-derived iPS cells (MIB2-6: Sci Rep, 2017) using the CRISPR/Cas9 system (MIB2-6 BG: Sci Rep, 2021). However, while MIB2-6 BG could differentiate into hematopoietic progenitor cells (HPC), they could not differentiate into CD19-positive (CD19+) cells when co-cultured with mouse stromal cells (MS-5) (Sci Rep, 2021). In this study, we performed co-culture of MIB2-6 BG and human mesenchymal stem cells (hMSCs: Tsutsumi S, BBRC, 2001) capable of differentiating umbilical cord blood into CD19+ cells. Additionally, we created MIB2-6 BG cells with p53 gene deficiency (MIB2-6 BG TP53KO), and examined whether they could differentiate into HPC and redifferentiate into B lymphocytes. Methods: After differentiation of MIB2-6BG cells into CD34-positive (CD34+) cells using the STEMdiffTM Hematopoietic Kit, they were purified using MACS CD34 microbeads, subjected to colony-forming (CF) assay using methylcellulose, and co-cultured with hMSCs in a medium containing SCF, Flt3-L, IL-3 and IL-6. Colony formation was assessed after 2 weeks, and floating cells were collected 2, 3, 4 and 5 weeks after co-culture and analyzed for cell surface antigens by flow cytometry (FCM) and CD19 and Pax5 expression by real-time PCR. We also conducted transplantation experiments to determine whether CD34+ cells derived from MIB2-6 BG and MIB2-6 BG TP53KO form tumors in NRG mice. Results:CD34+ cells derived from MIB2-6 BG and MIB2-6 BG TP53KO were able to differentiate into granulocytes, macrophages, and erythroblasts in the CF assay. Furthermore, the number of colonies increased significantly with MIB2-6 BG TP53KO. FCM revealed a small number of CD19weakly+/CD10+ cells 4 weeks after the start of co-culture of both MIB2-6 BG and MIB2-6 BG TP53KO with hMSCs, whereas real-time PCR demonstrated faint CD19 expression in CD34+ cells before co-culture, which increased 3 weeks after co-culture. Pax5 expression was detected in both the iPS cells, but could not be detected after co-culture with hMSCs. Interestingly, CD3+/CD4+/CD8- cells were observed 3 weeks after the start of co-culture of both MIB2-6 BG and MIB2-6 BG TP53KO with hMSCs. On the other hand, no tumor formation was observed in NRG mice 4 to 6 months after transplantation. Conclusion: Both MIB2-6 BG and MIB2-6 BG TP53KO cells were able to differentiate into CD34+ hematopoietic progenitor cells, with the additional potential to differentiate into lymphocytes. In addition, our results confirmed that the deletion of the p53 gene enhances the proliferation of CD34+cells, although further refinement is needed to create a tumorigenesis model using iPS cells derived from normal B lymphocytes.