The immunological synapse between T cells and tumor cells is believed to be important for effective tumor clearance. However, the immunological synapse has never been imaged or analyzed in detail in human tissue. In this work, intercellular interactions between T cells and tumor cells were analyzed in detail in human glioblastoma. After characterization of the population of infiltrating T cells by multiple immunofluorescence staining and stereological quantification, the microanatomy of T cell-tumor cell intercellular communication was analyzed in detail using confocal microscopy and three-dimensional rendering. Cytotoxic T lymphocytes that infiltrated human glioblastoma underwent rearrangement when in contact with tumor cells, to form a three-dimensional structure in the intercellular contact area; this was characterized by microclusters of the CD3/TCR complex, re-arrangement of the cytoskeleton, and granzyme B polarization. In addition, such T cell-targeted cells show fragmentation of the microtubular system and increased expression levels of cleaved caspase 3, which suggests that cytotoxic T lymphocytes likely provoke changes in tumor cells and subsequently induce cell death. These results show that the formation of the cytotoxic T lymphocyte immunological synapse occurs in human tissue and may be relevant for the effective immune-mediated clearance of tumorigenic cells, therefore opening up new avenues for glioblastoma immunotherapy.