The success of dendritic cell (DC)-based immunotherapeutics critically hinges on the capacity of the vascularly administered cells to enter tissues. Transendothelial migration (TEM) is dictated by an ordered cascade of receptor/ligand interactions. In this study, we examined the key molecular effectors of TEM of human monocyte-derived DCs (mo-DCs) generated by clinically relevant methods: CD14 selection (CD14-S) and plastic adherence selection (PA-S). Without chemokine input, CD14-S cells undergo greater TEM than PA-S cells over TNF-a-stimulated HUVECs. TEM of CD14-S mo-DCs is E-selectin/very late Ag-4 (VLA-4) dependent, and engagement of E-selectin ligands activates VLA-4 on CD14-S mo-DCs but not on PA-S mo-DCs. E-selectin binding glycoforms of P-selectin glycoprotein ligand-1 (PSGL-1) (i.e., cutaneous lymphocyte Ag [CLA]) and CD44 (i.e., hematopoietic cell E-selectin/L-selectin ligand [HCELL]) are both expressed on CD14-S mo-DCs, but only CLA is expressed on PA-S mo-DCs. To elucidate the effect of CD44 or PSGL-1 engagement, mo-DCs were pretreated with their ligands. Ligation of CD44 on CD14-S mo-DCs triggers VLA-4 activation and TEM, whereas PSGL-1 ligation does not. HCELL expression on CD14-S mo-DC can be enforced by cell surface exofucosylation, yielding increased TEM in vitro and enhanced extravasation into bone marrow in vivo. These findings highlight structural and functional pleiotropism of CD44 in priming TEM of mo-DCs and suggest that strategies to enforce HCELL expression may boost TEM of systemically administered CD14-S mo-DCs.