Upon recognition of cognate antigens via their TCR, CD8+ cytotoxic T lymphocytes eliminate infected or tumor cells through the immunological synapse (IS), a complex structure used to both decode signals and deliver cytotoxic molecules. Although co-stimulatory receptors are known to amplify TCR signaling, their ability to modulate IS assembly to sustain elimination of target cells has remained poorly explored. Given the diversity of co-stimulatory receptors and their distinct signaling modules, we hypothesized that these receptors might differentially affect IS assembly. In order to systematically compare the impact of co-stimulatory receptors engagement on various facets of IS architecture, we applied a high-content/high-throughput confocal microscopy analysis of the IS to pre-activated human CD8+ T cells co-expressing homogenous levels of ICOS, CD2, CD226, CD27, OX40 and GITR. Engagement of these co-stimulatory receptors, together with the TCR and LFA-1, promoted synaptic adhesion to varying degrees. Remarkably, each co-stimulatory receptor trigger imprinted a distinct IS architecture, as revealed by unsupervised dimensionality reduction analysis. In particular, the engagement of CD226, ICOS or CD2 enhanced IS spreading through the formation of unique types of actin-rich protrusions. Notably, CD226 stimulation promoted the formation of a high-affinity LFA-1 belt. Consistent with this IS patterning, CD226 increased T cell adherence to target cells and facilitated killing. Furthermore, co-stimulatory receptor engagement affected, to varying degrees, the polarization of the microtubule-organizing center (MTOC) and the distribution of lytic granules at the synaptic area. We therefore expect to decipher the molecular events underlying the specific IS modulatory effects of multiple co-stimulatory receptors, with potential impact for the field of immunotherapy.