Mixed phenotype acute leukemia (MPAL) is a rare and aggressive form of leukemia with a poor prognosis and no established treatment. In this study, we established a novel leukemic cell line, JMPAL-1, from a specimen of a 69-year-old patient with Philadelphia chromosome-positive MPAL. Flow cytometry showed that JMPAL-1 expresses B-cell markers but not myeloperoxidase. A genomic analysis of JMPAL-1 cells revealed the BCR::ABL1 fusion gene, missense mutation in PAX5, homozygous deletion of CDKN2A/CDKN2B, and BRAF amplification. This cell line was stroma-dependent in proliferation and required co-culturing with mouse bone marrow-derived mesenchymal cells (9-15C). Knowing the differences between JMPAL-1 and patient leukemia cells may improve understanding of the in vivo versus in vitro behavior of leukemia, clonal selection, and transformation. The stroma-dependent growth pattern of JMPAL-1 also provides a unique platform to study tumor-stromal interactions and their role in leukemic cell survival and drug resistance. Our study highlights the importance of establishing preclinical models such as JMPAL-1 and performing detailed cytogenetic analysis to develop targeted therapies in line with the pathogenesis of the disease.