Inversion of chromosome 16 [inv(16)] is one of the most common chromosomal rearrangements in Acute Myeloid Leukemia (AML) and generates the fusion gene CBFB::MYH11 (CM), which initiates leukemogenesis. Patients with inv(16) at diagnosis invariably have the rearrangement at relapse, leading to the assumption that CM is required after leukemic transformation. However, this has yet to be shown experimentally. Using a knock-in mouse that allows for deletion of CM after leukemia development, we found that loss of the fusion gene increased apoptosis and decreased colony growth in vitro. Interestingly, 5-20% of the colonies had successfully deleted CM. To test the role of CM in vivo, we used an inducible shRNA knockdown (KD) construct against the fusion gene. We found that decreased CM expression eliminated leukemia cells from the peripheral blood and spleen, but not the bone marrow, despite these cells showing significant knockdown of CM at the mRNA and protein levels. Furthermore, with prolonged KD of CM, ~40% of mice re-established disease while maintaining KD of the fusion gene. Our work indicates that CM is required by leukemia cells in the spleen and blood, but that cells in the bone marrow can survive and re-establish disease independent of the fusion protein.