Here, we characterize the in vivo migratory behavior of AML cells and their response to chemotherapy and CXCR4 antagonism, using high‐resolution 2‐photon and confocal intravital microscopy of mouse calvarium BM and the well‐established MLL‐AF9‐driven AML mouse model. This evidence concerns the gene MLLT3 and acute myeloid leukemia.