Summary. Background: Immune tolerance induction (ITI) is the only therapeutic approach that can eradicate factor VIII (FVIII) inhibitors in patients with hemophilia A. Predictors of ITI outcome are still debated, and the role of F8 gene mutations in this is not well established. Objectives: To investigate the relationship between F8 genotype and ITI outcome in patients with severe hemophilia A and high-responding inhibitors. Patients and Methods:F8 mutations were identified in 86 patients recruited as part of the Italian ITI registry (the PROFIT study). ITI outcome was centrally reviewed according to the following definitions: success (undetectable inhibitor and normal FVIII pharmacokinetics), partial success (inhibitor titer < 5 BU mL−1 and/or abnormal FVIII pharmacokinetics), and failure. Results:F8 mutations known to be associated with a high risk of inhibitor development (large deletions, inversions, nonsense mutations and splice site mutations) were found in 70 patients (81%); among these, the intron 22 inversion was present in 49 patients (57%). In 16 patients (19%) lower-risk F8 defects (small insertions/deletions and missense mutations) were identified. The latter group of patients showed a significantly higher ITI success rate than those carrying high-risk mutations [13/16 (81%) vs. 33/70 (47%); risk ratio 1.7, 95% confidence interval (CI) 1.1–2.1, P = 0.01]. On multivariate analysis, the mutation risk class remained a significant predictor of success [adjusted odds ratio (OR) 6.2, 95% CI 1.1–36.0, P = 0.04], as were inhibitor titer at ITI start (< 5 BU mL−1, OR 11.8, 95% CI 3.5–40.2, P < 0.001), and peak titer during ITI (< 100 BU mL−1, OR 11.4, 95% CI 3.2–40.8, P < 0.001). Conclusions: ITI success is influenced by F8 genotype. This knowledge should contribute to the stratification of prognosis, and to the clinical choices made for ITI in patients with high-responding inhibitors.