The reverse transmigration (RT) of tissue-resident dendritic cells (DCs) across lymphatic endothelia is prerequisite for the initiation of adaptive immune responses and might be regulated in a manner similar to diapedesis. Specifically, CD31 and CD99, which act as gatekeepers during diapedesis, might have a role in RT of DCs. We found that human lymphatic endothelial cells (LECs) and DCs in vitro and in human skin explants express CD31 and CD99. In human skin, CD31 was enriched along intercellular surfaces of LECs, whereas CD99 was preferentially confined to luminal surfaces as evidenced by immunoelectron microscopy. Confocal microscopy analysis revealed that tumor necrosis factor-alpha (TNF-α) and CXCL12 acted as inducers of RT in vitro, but only CXCL12 stimulation resulted in a significant increase in migration rate of DCs. Upon TNF-α stimulation, CXCL12 mRNA levels transiently increased in human fibroblasts and LECs, whereas CXCL12 protein expression levels did not significantly change. Blocking mAbs to CD31 and CD99 significantly reduced RT of DCs across cultured human LEC monolayers and blocked CXCL12-induced migration of DCs in whole-skin explants. In sum, this study shows that CD31 and CD99 are involved in the RT of DCs across LECs and that similar mechanisms promote both diapedesis and RT.