HIV Tat can enhance activation-induced up-regulation of Fas ligand (FasL), which may contribute to T cell apoptosis in human immune deficiency virus (HIV)-infected individuals. We have assessed functional and physical interactions between Tat and the Egr family of transcription factors (Egr-1, -2, and -3), the latter two of which are major participants in activation-induced FasL up-regulation. Here we report that whereas Tat itself has no effect on the FasL promoter, it binds to Egr-2 and -3 and synergizes with them to superinduce expression of a FasL promoter-driven reporter. A Tat molecule containing a single amino acid substitution that results in the loss of transactivation activity for the HIV long terminal repeat still binds Egr-3 but can no longer enhance Egr-mediated transactivation of the FasL promoter. Furthermore, the mutated Tat acts as a dominant negative inhibitor, blocking the superinduction of FasL caused by wild type Tat. Because Tat is present in virus-infected cells and in the serum of HIV-infected individuals, these results suggest that increased expression of FasL in these circumstances may result from the cooperative activities of activation-induced Egrs and Tat.