AbstractEfficient bone marrow (BM) homing is a prerequisite for successful engraftment of transplanted hematopoietic cells (HPCs). Contradictory conclusions about the contribution of SDF-1/CXCR4 have clouded our understanding of its role within the molecular pathway cooperation needed for BM homing, particularly with the well-defined hierarchic network of adhesion molecules. In the present study we sought to unravel cooperative and compensatory molecular pathways guiding BM homing. Fresh BM-HPCs, rendered either SDF-1 unresponsive or Gi-signaling refractory, homed quite efficiently, because of compensation by α4-integrin interacting with VCAM-1. The contribution of SDF-1/CXCR4- or Gi-protein-mediated signals to BM homing became apparent after their blockade was combined with deletion of α4-integrin, leading to dramatic reduction in BM homing. Similar conclusions were revealed when VCAM-1-deficient hosts were used. Cytokine incubation changed the functional properties of BM-HPCs and hierarchy of molecular pathway usage in homing, by shifting the dominance among the homing mediators: loss of CXCR4 or Gi-signaling now significantly reduced BM homing, with only partial compensation through α4/VCAM-1 and endothelial selectins. These studies depict a flexible hierarchy of cooperating homing pathways, in which dominant players are repositioned with changing cytokine milieu, and possibly source of HPCs.