Killer T cells (cytotoxic T lymphocytes, CTLs) are responsible for maintaining immune homoeostasis by eliminating virus-infected and cancerous cells. CTLs bind to, and form an immunological synapse with their targets, then secrete a Ca2+-dependent pore-forming protein (perforin) and a cocktail of pro-apoptotic serine proteases (granzymes) into the synaptic cleft. Although the CTL and the target cell plasma membranes are both exposed to perforin within the synapse, only the target cell membrane is disrupted. As a consequence, granzymes will penetrate and kill the target cell, while the CTL is invariably spared. What governs CTL resistance to its own secreted perforin is unknown. Here, we report that CTLs achieve this via the physical properties of their plasma membrane within the synapse. We identify two protective mechanisms: the CTL membrane repels perforin due to its high lipid order and, in addition, it exposes phosphatidylserine within the synapse, which by its negative charge sequesters and inactivates perforin. The resulting resistance of CTLs to perforin explains their ability to kill target cells in rapid succession and to survive these encounters, thus enabling CTLs to maintain immune homoeostasis. Furthermore, these mechanisms imply a hitherto unsuspected role for plasma membrane lipid organization in protecting cells from immune attack.