In this study, optical techniques were used to characterize adhesion of hematopoietic cells to bone marrow (BM) stromal microenvironment in situ. Bone marrow cells (BMC) labeled with PKH membrane linkers were infused into nonconditioned femurs and were monitored by fluorescence microscopy through an optical bone window. Repeated infusions of BMC into the femoral lumen resulted in a progressive increase in the number of adherent cells (p < 0.01), indicating that the availability of hemopoietic niches in the nonconditioned BM was not a rate-limiting factor of early BMC seeding. Adhesion of hemopoietic progenitor and stem cells (HSPC) was 30-fold higher than lineage(+) BMC (p < 0.001), suggesting that adhesion molecules on the surface of HSPC have a higher propensity for adhesion. BMC antigen-matched to and disparate from BM stroma adhered at equal rates, opposing the idea of involvement of antigen barriers during early seeding. It is concluded that primary adhesion to BM stromal microenvironment is favorable for HSPC and is not restricted by antigenic barriers or availability of vacant niches.