Phosphatase and tensin homologue (PTEN) loss and activation of the Akt-mammalian target of rapamycin (mTOR) pathway increases mRNA translation, increases levels of the antiapoptotic protein FLIP(S), and confers resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in glioblastoma multiforme (GBM). In PTEN-deficient GBM cells, however, the FLIP(S) protein also exhibited a longer half-life than in PTEN mutant GBM cells, and this longer half-life correlated with decreased FLIP(S) polyubiquitination. FLIP(S) half-life in PTEN mutant GBM cells was reduced by exposure to an Akt inhibitor, but not to rapamycin, suggesting the existence of a previously undescribed, mTOR-independent linkage between PTEN and the ubiquitin-dependent control of protein stability. Total levels of the candidate FLIP(S) E3 ubiquitin ligase atrophin-interacting protein 4 (AIP4) were comparable in PTEN wild-type (WT) and PTEN mutant GBM cells, although in PTEN-deficient cells, AIP4 was maintained in a stable polyubiquitinated state that was less able to associate with FLIP(S) or with the FLIP(S)-containing death inducing signal complex. Small interfering RNA-mediated suppression of AIP4 levels in PTEN WT cells decreased FLIP(S) ubiquitination, prolonged FLIP(S) half-life, and increased TRAIL resistance. Similarly, the Akt activation that was previously shown to increase TRAIL resistance did not alter AIP4 levels, but increased AIP4 ubiquitination, increased FLIP(S) steady-state levels, and suppressed FLIP(S) ubiquitination. These results define the PTEN-Akt-AIP4 pathway as a key regulator of FLIP(S) ubiquitination, FLIP(S) stability, and TRAIL sensitivity and also define a novel link between PTEN and the ubiquitin-mediated control of protein stability.