Major features of the transcellular signaling mechanism responsible for endothelium-dependent regulation of vascular smooth muscle tone are unresolved. We identified local calcium (Ca²⁺) signals (“sparklets”) in the vascular endothelium of resistance arteries that represent Ca²⁺ influx through single TRPV4 cation channels. Gating of individual TRPV4 channels within a four-channel cluster was cooperative, with activation of as few as three channels per cell causing maximal dilation through activation of endothelial cell intermediate (IK)- and small (SK)-conductance, Ca²⁺-sensitive potassium (K⁺) channels. Endothelial-dependent muscarinic receptor signaling also acted largely through TRPV4 sparklet-mediated stimulation of IK and SK channels to promote vasodilation. These results support the concept that Ca²⁺ influx through single TRPV4 channels is leveraged by the amplifier effect of cooperative channel gating and the high Ca²⁺ sensitivity of IK and SK channels to cause vasodilation.