To study the role of bone marrow endothelial cells (BMEC) in the regulation of hematopoietic cell trafficking, we have designed an in vitro model of transendothelial migration of hematopoietic progenitor cells and their progeny. For these studies, we have taken advantage of a human BMEC-derived cell line (BMEC-1), which proliferates independent of growth factors, is contact inhibited, and expresses adhesion molecules similar to BMEC in vivo. BMEC-1 monolayers were grown to confluency on 3 μm microporous membrane inserts and placed in 6-well tissue culture plates. Granulocyte-colony stimulating factor (G-CSF )–mobilized peripheral blood CD34⁺ cells were added to the BMEC-1 monolayer in the upper chamber of the 6-well plate. After 24 hours of coincubation, the majority of CD34⁺ cells remained nonadherent in the upper chamber, while 1.6 ± 0.3% of the progenitor cells had transmigrated. Transmigrated CD34 cells expressed a higher level of CD38 compared with nonmigrating CD34⁺ cells and may therefore represent predominantly committed progenitor cells. Accordingly, the total plating efficiency of the transmigrated CD34⁺ cells for lineage-committed progenitors was higher (14.0 ± 0.1 v 7.8% ± 1.5%). In particular, the plating efficiency of transmigrated cells for erythroid progenitors was 27-fold greater compared with nonmigrating cells (8.0% ± 0.8% v 0.3% ± 0.1%) and 5.5-fold compared with unprocessed CD34⁺ cells (2.2% ± 0.4%). While no difference in the expression of the β1-integrin very late activation antigen (VLA)-4 and β2-integrin lymphocyte function-associated antigen (LFA)-1 was found, L-selectin expression on transmigrated CD34⁺ cells was lost, suggesting that shedding had occurred during migration. The number of transmigrated cells was reduced by blocking antibodies to LFA-1, while L-selectin and VLA-4 antibodies had no inhibitory effect. Continuous coculture of the remaining CD34⁺ cells in the upper chamber of the transwell inserts resulted in proliferation and differentiation into myeloid and megakaryocytic cells. While the majority of cells in the upper chamber comprised proliferating myeloid precursors such as promyelocytes and myelocytes, only mature monocytes and granulocytes were detected in the lower chamber. In conclusion, BMEC-1 cells support transmigration of hematopoietic progenitors and mature hematopoietic cells. Therefore, this model may be used to study mechanisms involved in mobilization and homing of CD34⁺ cells during peripheral blood progenitor cell transplantation and trafficking of mature hematopoietic cells.