The t(10;11)(p13-14;q14-21) translocation, giving rise to the CALM-AF10 fusion gene, is a recurrent chromosomal rearrangement observed in patients with poor prognosis acute myeloid leukemia (AML). Although splicing of the CALM-AF10 fusion transcripts has been described in AML patients, the contribution of different CALM and AF10 domains to in vivo leukemogenesis remains to be defined. We therefore performed detailed structure-function studies of the CALM-AF10 fusion protein. We demonstrate that fusion of the C-terminal 248 amino acids of CALM, which include the clathrin-binding domain, to the octapeptide motif-leucine-zipper (OM-LZ) domain of AF10 generated a fusion protein (termed CALM-AF10 minimal fusion (MF)), with strikingly enhanced transformation capabilities in colony assays, providing an efficient system for the expeditious assessment of CALM-AF10-mediated transformation. Leukemias induced by the CALM-AF10 (MF) mutant recapitulated multiple aspects of full-length CALM-AF10-induced leukemia, including aberrant Hoxa cluster upregulation, a characteristic molecular lesion of CALM-AF10 leukemias. In summary, this study indicates that collaboration of the clathrin-binding and the OM-LZ domains of CALM-AF10 is sufficient to induce AML. These findings further suggest that future approaches to antagonize CALM-AF10-induced transformation should incorporate strategies, which aim at blocking these key domains.