Activated vascular endothelial cells (ECs) express major histocompatibility complex (MHC) class II molecules in vitro and in vivo in acute and chronic allograft rejection. However, human ECs may be limited in their ability to effectively activate CD4⁺ T cells, because they do not express members of the B7 family (CD80 and CD86) of costimulatory molecules. In this study, we show that ECs promote the full activation of CD4⁺ T cells via trans-costimulatory interactions. By reverse transcriptase polymerase chain reaction, Western blot, and FACS^® analysis, we could not detect the expression of CD80 and CD86 on activated ECs and found minimal expression on purified CD4⁺ T cells. In contrast, both CD80 and CD86 were expressed in allogeneic CD4⁺ T cell–EC cocultures. Expression of CD86 peaked at early times between 12 and 24 h after coculture, whereas CD80 was not expressed until 72 h. Addition of anti-CD86 but not anti-CD80 monoclonal antibodies to cocultures inhibited IL-2 production and the proliferation of CD4⁺ T cells to allogeneic donor human umbilical vein ECs (HUVECs), as well as to skin and lung microvascular ECs. Furthermore, we found that interferon γ–activated ECs but not untreated ECs induced mRNA and cell surface expression of CD80 and CD86 on CD4⁺ T cells, and these T cells were functional to provide a trans-costimulatory signal to autologous CD4⁺ T cells. Blockade of MHC class II and lymphocyte function–associated antigen 3 but not other EC cell surface molecules on IFN-γ–activated ECs inhibited the induction of CD86 on CD4⁺ T cells. Transmigration of purified populations of monocytes across EC monolayers similarly resulted in the induction of functional CD86, but also induced the de novo expression of the cytokines interleukin (IL)-1α and IL-12. In addition, EC-modified monocytes supported enhanced proliferation of allogeneic and autologous CD4⁺ T cells. Taken together, these data define the ability of the endothelium to modify CD4⁺ T cells and monocytes for trans-costimulatory events. This unique function of the endothelium in alloimmune T cell activation has functional consequences for the direct and the indirect pathways of allorecognition.