Few techniques are available to characterize in vivo the early cellular dynamics of long-term reconstitution of hematopoiesis after transplantation of hematopoietic stem cells (HSCs) after lethal irradiation. Using a fiber-optic imaging system, we track the early steps of in vivo recruitment and proliferation of Lin⁻Sca-1⁺c-Kit⁺CD34⁻ (LSKCD34⁻) HSCs highly enriched in HSCs and transplanted into lethally irradiated mice. Recruitment of the transplanted LSKCD34⁻ hematopoietic cells first occurs in the femoral head and is continuous during 24 hours. Quantification of the fluorescence emitted by the transplanted hematopoietic cells shows that proliferation of LSKCD34⁻ hematopoietic cells in the femoral head was potent 3 days after transplantation. Using a development of this fiber-optic imaging system, we show that the transplanted LSKCD34⁻ hematopoietic cells are associated with vascularized structures as early as 5 hours after transplantation. This early association is dependent on reactive oxygen species (ROS) partly through the regulation of vascular cell adhesion molecule-1 expression on endothelial cells and is followed by a ROS-dependent proliferation of LSKCD34⁻ hematopoietic cells. This new in vivo imaging technique permits the observation of the early steps of hematopoietic reconstitution by HSCs in long bones and shows a new role of ROS in the recruitment of HSCs by bone marrow endothelial cells.