The cytotoxic function of CD178 (Fas ligand (FasL)) is critical to the maintenance of peripheral tolerance and immune-mediated tissue pathology. The active site of FasL resides at the FasL extracellular region (FasL(Ext)) and it functions through binding/cross-linking Fas receptor on target cells. In this study, we report that FasL(Ext)-mediated cytotoxicity is regulated by the FasL cytoplasmic tail (FasL(Cyt)). Deleting the N-terminal 2-70 aa (delta70) or N-terminal 2-33 aa (delta33) reduced the cytotoxic strength as much as 30- to 100-fold. By contrast, change in the cytotoxic strength was not observed with FasL deleted of the proline-rich domains (45-74 aa, delta PRD) in the FasL(Cyt). Our study identifies a novel function of FasL(Cyt) and demonstrates that FasL(2-33), a sequence unique to FasL, is critically required for the optimal expression of FasL(Ext)-mediated cytotoxicity.