Proper T cell activation is promoted by sustained calcium signaling downstream of the TCR. However, the dynamics of calcium flux after stimulation with an APC in vivo remain to be fully understood. Previous studies focusing on T cell motility suggested that the activation of naive T cells in the lymph node occurs in distinct phases. In phase I, T cells make multiple transient contacts with dendritic cells before entering a phase II, where they exist in stable clusters with dendritic cells. It has been suggested that T cells signal during transient contacts of phase I, but this has never been shown directly. Because time-dependent loss of calcium dyes from cells hampers long-term imaging of cells in vivo after antigenic stimulation, we generated a knock-in mouse expressing a modified form of the Cameleon fluorescence resonance energy transfer reporter for intracellular calcium and examined calcium flux both in vitro and in situ. In vitro, we observed transient, oscillatory, and sustained calcium flux after contact with APC, but these behaviors were not affected by the type of APC or Ag quantity, but were, however, moderately dependent on Ag quality. In vivo, we found that during phase I, T cells exhibit weak calcium fluxes and detectable changes in cell motility. This demonstrates that naive T cells signal during phase I and support the hypothesis that accumulated calcium signals are required to signal the beginning of phase II.