The mechanism by which hematopoietic stem and progenitor cells (HSPCs) through interaction with their niches maintain and reconstitute adult hematopoietic cells is unknown. To functionally and genetically track localization of HSPCs with their niches, we employed novel mutant loxPs, lox66 and lox71 and Cre-recombinase technology to conditionally delete c-Kit in adult mice, while simultaneously enabling GFP expression in the c-Kit-deficient cells. Conditional deletion of c-Kit resulted in hematopoietic failure and splenic atrophy both at steady state and after marrow ablation leading to the demise of the treated adult mice. Within the marrow, the c-Kit-expressing GFP + cells were positioned to Kit ligand (KL)-expressing niche cells. This c-Kit-mediated cellular adhesion was essential for long-term maintenance and expansion of HSPCs. These results lay the foundation for delivering KL within specific niches to maintain and restore hematopoiesis.