Perforin is an immune effector used by cytotoxic T lymphocytes (CTLs) to perforate the plasma membrane of target cells and introduce pro-apoptotic granzymes. Using a combination of atomic force and electron microscopy (AFM/EM) on artificial lipid membranes, we followed perforin oligomerization and membrane insertion at the nanoscale and uncovered a novel pore forming mechanism. However, CTLs employing perforin only disrupt the target cell membrane, and how CTLs escape unscathed remained a mystery. We thus extended our AFM/EM approach to more complex artificial lipid membranes and used immunological assays to study perforin behaviour on live cells. We found two protective properties of the CTL plasma membrane: high lipid order repels most perforin and, in addition, exposed phosphatidylserine sequesters and inactivates the remaining protein. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters.