BACKGROUND: Many diseases associated with bone marrow transplantation (BMT) are caused by transplanted hematopoietic cells, and the onset of these diseases occurs after homing of donor cells in the initial phase after BMT. Noninvasive observation of donor cell homing shortly after transplantation is potentially valuable for improving therapeutic outcomes of BMT by diagnosing the early stages of these diseases. METHODOLOGY/PRINCIPAL FINDINGS: Freshly harvested near-infrared fluorescence-labeled cells were noninvasively observed for 24 h after BMT using a photon counting device to track their homing process. In a congenic BMT model, the homing of Alexa Fluor 750-labeled donor cells in the tibia was detected less than 1 h after BMT. In addition, subsequent cell distribution in an intraBM BMT model was successfully monitored for the first time using this method. In the allogeneic BMT model, T-cell depletion decreased the near-infrared fluorescence (NIRF) signals of the reticuloendothelial system. CONCLUSIONS/SIGNIFICANCE: This approach in several murine BMT models revealed that the transplanted cells homed within 24 h after transplantation. NIRF labeling is useful for tracking transplanted cells in the initial phase after BMT, and this approach can contribute to in vivo studies aimed at improving the therapeutic outcomes of BMT.