Hematopoietic stem cells (HSCs) engage in complex bidirectional signals with the hematopoietic microenvironment (HM), and there is emerging evidence that leukemia stem cells (LSCs) may use similar interactions. Using a syngeneic retroviral model of MLL-AF9 induced acute myeloid leukemia (AML), we have identified 2 different stages of leukemia progression, propagated by "pre-LSCs" and established leukemia (LSCs) and compared the homing properties of these distinctive entities to that of normal HSCs. The homing and microlocalization of pre-LSCs was most similar to long-term HSCs and was dependent on cell-intrinsic Wnt signaling. In contrast, the homing of established LSCs was most similar to that of committed myeloid progenitors and distinct from HSCs. Although osteoblast-derived Dickkopf-1, a potent Wnt inhibitor known to impair HSC function, dramatically impaired normal HSC localization within the bone marrow, it did not affect pre-LSCs, LSC homing, or AML development. Mechanistically, cell-intrinsic Wnt activation was observed in human and murineAMLsamples, explaining the independence of MLL-AF9 LSCs from nichederived Wnt signals. These data identify differential engagement of HM associated with leukemic progression and identify an LSC niche that is physically distinct and independent of the constraints of Wnt signaling that apply to normal HSCs.