The CALM/AF10 fusion gene is found in various hematological malignancies including AML, T-cell ALL, and malignant lymphoma. We have previously identified the leukemia stem cell (LSC) in a CALM/AF10-driven murine bone marrow transplant acute myeloid leukemia model as B220-positive lymphoid cells with B cell characteristics. To identify the target cell for leukemic transformation or 'cell of origin of leukemia' (COL) in non-disturbed steady-state hematopoiesis we inserted the CALM/AF10 fusions gene preceded by a loxP-flanked transcriptional stop cassette into the Rosa26 locus. Vav-Cre induced pan-hematopoietic expression of the CALM/AF10 fusion gene led to acute leukemia with a median latency of 12 months. Mice expressing CALM/AF10 in the B lymphoid compartment using Mb1-Cre or CD19-Cre inducer lines did not develop leukemia. Leukemias had a predominantly myeloid phenotype but showed co-expression of the B cell marker B220, and had clonal B-cell receptor rearrangements. Using whole-exome sequencing, we identified an average of 2 to 3 additional mutations per leukemia, including activating mutations in known oncogenes like FLT3 and PTPN11. Our results show that the COL for CALM/AF10 is a stem or early progenitor cell and not a cell of B-cell lineage with a phenotype similar to that of the LSC in CALM/AF10-positive leukemia.