Cytotoxic T cell killing is executed at the immunological synapse, whose nanoscale organization underlies function but remains difficult to resolve in native states. Here, we apply cryo-expansion microscopy (cryo-ExM) to visualize the near-native three-dimensional architecture of human T cell synapses and cytotoxic organelles. Cryo-ExM preserves actin, microtubules, membranes, and fine membrane protrusions with high fidelity, enabling volumetric quantification of synapse morphogenesis. We identify an adhesion-dependent, dome-like membrane architecture beneath activated T cells that collapses upon ICAM-1 engagement, linking synapse topology to adhesive cues. Cryo-ExM further resolves intact lytic granules in primary human CD4 and CD8 T cells, revealing single-core and multi-core ultrastructures, spatial organization, and perforin and granzyme loading. Using tissue-adapted expansion microscopy, we map cytotoxic granule content in tumor-infiltrating T cells in FFPE human brain tumors. Together, these data establish a near-native structural framework for human T cell cytotoxicity and an imaging workflow bridging cell models and clinical tissues.