Ca²⁺-release-activated Ca²⁺ (CRAC) channels generate sustained Ca²⁺ signals that are essential for a range of cell functions, including antigen-stimulated T lymphocyte activation and proliferation^,. Recent studies have revealed that the depletion of Ca²⁺ from the endoplasmic reticulum (ER) triggers the oligomerization of stromal interaction molecule 1 (STIM1), the ER Ca²⁺ sensor, and its redistribution to ER–plasma membrane (ER–PM) junctions^,,,, where the CRAC channel subunit ORAI1 accumulates in the plasma membrane and CRAC channels open^,,,. However, how the loss of ER Ca²⁺ sets into motion these coordinated molecular rearrangements remains unclear. Here we define the relationships among [Ca²⁺]_ER, STIM1 redistribution and CRAC channel activation and identify STIM1 oligomerization as the critical [Ca²⁺]_ER-dependent event that drives store-operated Ca²⁺ entry. In human Jurkat leukaemic T cells expressing an ER-targeted Ca²⁺ indicator, CRAC channel activation and STIM1 redistribution follow the same function of [Ca²⁺]_ER, reaching half-maximum at ∼200 µM with a Hill coefficient of ∼4. Because STIM1 binds only a single Ca²⁺ ion, the high apparent cooperativity suggests that STIM1 must first oligomerize to enable its accumulation at ER–PM junctions. To assess directly the causal role of STIM1 oligomerization in store-operated Ca²⁺ entry, we replaced the luminal Ca²⁺-sensing domain of STIM1 with the 12-kDa FK506- and rapamycin-binding protein (FKBP12, also known as FKBP1A) or the FKBP-rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR, also known as FRAP1). A rapamycin analogue oligomerizes the fusion proteins and causes them to accumulate at ER–PM junctions and activate CRAC channels without depleting Ca²⁺ from the ER. Thus, STIM1 oligomerization is the critical transduction event through which Ca²⁺ store depletion controls store-operated Ca²⁺ entry, acting as a switch that triggers the self-organization and activation of STIM1–ORAI1 clusters at ER–PM junctions.