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. Here, CXCR4 is linked to acute myeloid leukemia.