Cytotoxic T lymphocytes (CTLs) specialise in killing virus-infected and cancerous cells in our body. They do so by exposing targets cells to pro-apoptotic granzymes, whose entrance into the target is facilitated by the pore-forming protein perforin. Interestingly, while CTLs are exposed to this toxic protein during synapse formation they remain almost invariably unscathed, allowing them to kill multiple target cells in succession. This resistance stems from perforin’s unidirectional action, recently demonstrated to arise from a dynamic control of the CTL membrane lipid composition. Firstly, the CTL membrane inhibits perforin binding via its high plasma membrane order and secondly, exposed phosphatidylserine (PS) sequesters any residual perforin and inactivates it. This project’s first aim is to employ in vivo, in vitro and in silico methods to elucidate the spatio-temporal hierarchy of this two-fold defence mechanism in immunological synapses. Super-resolution (Lattice Light Sheet and Total Internal Reflection) microscopy studies will be conducted to elucidate the spatio-temporal interplay of PS exposure and enhanced membrane lipid order with events leading to synapse formation and target-cell killing. Secondly, a Brownian dynamics model will be developed to study the pathway for perforin nanopore formation. Research into these mechanisms holds unprecedented medical relevance, given how important successive killing by CTLs is to viral and cancer clearance. Finally, a better understanding of the pathway of perforin nanopore assembly could guide future therapeutic studies in locating targets to suppress or promote immune response.