Abstract Purpose: Artificial antigen-presenting cells, aAPC, have successfully been used to stimulate antigen-specific T-cell responses in vitro as well as in vivo. Although aAPC compare favorably with autologous dendritic cells in vitro, their effect in vivo might be diminished through rapid clearance by macrophages. Therefore, to prevent uptake and minimize clearance of aAPC by macrophages, thereby increasing in vivo functionality, we investigated the efficiency of “don't eat me” three-signal aAPC compared with classical two-signal aAPC. Experimental Design: To generate “don't eat me” aAPC, CD47 was additionally immobilized onto classical aAPC (aAPCCD47+). aAPC and aAPCCD47+ were analyzed in in vitro human primary T-cell and macrophage cocultures. In vivo efficiency was compared in a NOD/SCID T-cell proliferation and a B16-SIY melanoma model. Results: This study demonstrates that aAPCCD47+ in coculture with human macrophages show a CD47 concentration–dependent inhibition of phagocytosis, whereas their ability to generate and expand antigen-specific T cells was not affected. Furthermore, aAPCCD47+-generated T cells displayed equivalent killing abilities and polyfunctionality when compared with aAPC-generated T cells. In addition, in vivo studies demonstrated an enhanced stimulatory capacity and tumor inhibition of aAPCCD47+ over normal aAPC in conjunction with diverging biodistribution in different organs. Conclusions: Our data for the first time show that aAPC functionalized with CD47 maintain their stimulatory capacity in vitro and demonstrate enhanced in vivo efficiency. Thus, these next-generation aAPCCD47+ have a unique potential to enhance the application of the aAPC technology for future immunotherapy approaches. Clin Cancer Res; 21(9); 2075–83. ©2015 AACR.