Optimal T cell activation requires Ag recognition through the TCR， engagement of costimulatory molecules， and cytokines. T cells can also directly recognize danger signals through the expression of TLRs. Whether TLR ligands have the capacity to provide costimulatory signals and enhance Ag-driven T cell activation is not well understood. In this study， we show that TLR2 and TLR7 ligands potently lower the Ag threshold for cytokine production in T cells. To investigate how TLR triggering supports cytokine production， we adapted the protocol for flow cytometry-based fluorescence in situ hybridization to mouse T cells. The simultaneous detection of cytokine mRNA and protein with single-cell resolution revealed that TLR triggering primarily drives de novo mRNA transcription. mRNA stabilization only occurs when the TCR is engaged. TLR2-， but not TLR7-mediated costimulation， can enhance mRNA stability at low Ag levels. Importantly， TLR2 costimulation increases the percentage of polyfunctional T cells， a hallmark of potent T cell responses. In conclusion， TLR-mediated costimulation effectively potentiates T cell effector function to suboptimal Ag levels.