The anti-apoptotic molecule FLIP³ (Fas-associated death domain-like IL (interleukin)-1-converting enzyme inhibitory protein) inhibits Fas-mediated apoptosis by blocking activation of caspase-8. We previously showed that expression of transgenic FLIP on thyroid epithelial cells (TEC) of DBA/1 and CBA/J mice promoted earlier resolution of granulomatous experimental autoimmune thyroiditis (G-EAT) in vivo. This study was undertaken to directly determine if transgenic FLIP expressed on cultured TEC can protect TEC from Fas-mediated apoptosis in vitro. The results indicate that cultured TEC from DBA/1 and CBA/J mice can be sensitized in vitro by IFN (interferon)- and TNF (tumor necrosis factor)- to undergo Fas-mediated apoptosis. Transgenic overexpression of FLIP protected cultured TEC of FLIP Tg+ DBA/1 and CBA/J mice from Fas-mediated apoptosis, and FLIP small interfering RNA (siRNA) transfection of cultured TEC of FLIP Tg+ DBA/1 and CBA/J mice abolished the protective effect. These in vitro results are consistent with our previous in vivo studies using DBA/1 and CBA/J FLIP Tg+ mice and provide direct support for the hypothesis that transgenic expression of FLIP promotes resolution of G-EAT by protecting TEC from apoptosis.