Initial T cell activation is triggered by the formation of highly dynamic, spatiotemporally restricted Ca²⁺microdomains. Purinergic signaling is known to be involved in Ca²⁺influx in T cells at later stages compared to the initial microdomain formation. Using a high-resolution Ca²⁺live-cell imaging system, we show that the two purinergic cation channels P2X4 and P2X7 not only are involved in the global Ca²⁺signals but also promote initial Ca²⁺microdomains tens of milliseconds after T cell stimulation. These Ca²⁺microdomains were significantly decreased in T cells fromP2rx4^−/−andP2rx7^−/−mice or by pharmacological inhibition or blocking. Furthermore, we show a pannexin-1–dependent activation of P2X4 in the absence of T cell receptor/CD3 stimulation. Subsequently, upon T cell receptor/CD3 stimulation, ATP release is increased and autocrine activation of both P2X4 and P2X7 then amplifies initial Ca²⁺microdomains already in the first second of T cell activation.