SLP-76 and Vav, two hematopoietic cell specific molecules, are critical for T cell development and activation. Following T cell antigen receptor stimulation, SLP-76 and Vav both undergo tyrosine phosphorylation and associate with each other via the SH2 domain of Vav and phosphorylated tyrosines of SLP-76. Furthermore, SLP-76 and Vav have a synergistic effect on interleukin (IL)-2 promoter activity in T cells. In this report, we show that two tyrosines, Tyr-113 and Tyr-128, of SLP-76 are required for its binding to Vav, both in vitro and in intact cells. Surprisingly, we find also that the interaction between SLP-76 and Vav is not required for their cooperation in augmenting IL-2 promoter activity, as the two molecules appear to function in different signaling pathways upstream of IL-2 gene expression. Overexpression of SLP-76 in the Jurkat T cell line potentiates the activities of both nuclear factor of activated T cells and AP-1 transcription factors. In contrast, overexpression of Vav leads to enhanced nuclear factor of activated T cells activity without affecting AP-1. Additionally, overexpression of Vav, but not SLP-76, augments CD28-induced IL-2 promoter activity. These findings suggest that the synergy between SLP-76 and Vav in regulating IL-2 gene expression reflects the cooperation between different signaling pathways.