Objective The BCR-ABL mutation, T315I, is a common mutation and is resistant to both imatinib and second-generation Abl kinase inhibitors. Although strategies to overcome resistance-mediated T315I mutation may improve the survival of BCR-ABL−positive leukemia patients, there is little information on cell-based studies. Materials and Methods We established a new human BCR-ABL−positive acute lymphoblastic leukemia (ALL) cell line, SK-9 with the T315I mutation, from the peripheral blood of a 36-year-old female patient. Results Growth kinetic studies revealed an approximate population doubling time of 48 hours. The common B-cell phenotype is a feature of the SK-9 cell line. Cells have the Philadelphia chromosome (Ph) with many structural abnormalities, as well as the T315I mutation in the BCR-ABL gene. Insertion of SK-9 cells into athymic nude mice induced the formation of tumors in the lymph node that infiltrated into the spleen and bone marrow. We examined the drug sensitivity of imatinib, dasatinib, and nilotinib using a cell proliferation assay and an immunoblot assay. Cell proliferation did not decrease after imatinib, dasatinib, or nilotinib treatment as compared to the BCR-ABL−positive chronic myeloid leukemia cell line K562. Because phosphorylation of BCR-ABL and Crk-L did not decrease after imatinib and dasatinib treatment, it is suggested that SK-9 is resistant to imatinib, dasatinib, and nilotinib. Conclusion This cell line may provide a useful model for in vitro and in vivo cellular and molecular studies of BCR-ABL−positive ALL with T315I mutation.