Extracellular nucleotides inhibit homing and engraftment of AML cells in immunodeficient mice. Low-density mononuclear cells (10 × 10⁶) from the PB or the BM of 5 newly diagnosed AML patients were injected into sublethally irradiated NSG-immunodeficient mice after overnight incubation with or without nucleotides or P2YR analogs. Mice were killed 16 hours later to evaluate the homing of human cells. Nucleated cells obtained from the BM of long bones were stained and analyzed by flow cytometry for the detection of human CD45⁺ cells, as described in “Methods.” The homing capacity of AML cells to the BM was significantly inhibited by ATP (A). Similarly, when the long-term engraftment of leukemic cells pretreated with nucleotides was analyzed, we found a significant inhibition by ATP and by UTP (B). Furthermore, CD34⁺CD38⁻ cells from 5 AML patients at diagnosis were sorted and analyzed for the presence of the same molecular marker found in the unselected blast population. The results shown in panel C confirmed the leukemic origin of the study cell populations. Purified CD34⁺CD38⁻ LSCs preincubated with or without nucleotides were injected into NSG-immunodeficient mice, and a significant inhibition of long-term engraftment of leukemic hematopoiesis was observed under all the tested experimental conditions (D). *P < .05.