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Objective. After transplantation of hematopoietic stem cells, adhesion molecules play a major role in the multistep process of engraftment in which L-selectin is suggested to be of relevance. A positive correlation previously was found between the number of reinfused L-selectin+ stem cells and platelet recovery. In the present study, we determined the role of L-selectin in different engraftment steps, i.e., adhesion to endothelial cells, migration, and clonogenic outgrowth by in vitro assays that closely mimic the in vivo situation. Materials and Methods. Flow adhesion and migration experiments were performed using the human bone marrow endothelial cell line 4LHBMEC and isolated peripheral CD34+ cells with or without blocking of L-selectin-ligand interaction. Various clonogenic assays, including serum-free colony-forming unit-megakaryocytes (CFU-MK) and burst-forming unit-megakaryocytes (BFU-MK), were performed with sorted L-selectin+L-selectin- cells or in the presence of antibodies. Results. Blocking of L-selectin on CD34+ cells did not significantly affect rolling over and firm adhesion to 4LHBMEC. In addition, no role for L-selectin was found in transendothelial migration experiments. Finally, in clonogenic outgrowth of sorted or anti-L-selectin monoclonal antibody-incubated CD34+ cells, no key role for L-selectin expression could be defined in BFU-MK and CFU-MK assays. Conclusion. Using in vitro assays for CD34+ stem cell adhesion, migration, and clonogenic capacity, we were not able to define a major role for L-selectin. © 2002 International Society for Experimental Hematology. Published by Elsevier Science Inc.