CheckMate 568 is an open-label phase II trial that evaluated the efficacy and safety of nivolumab plus low-dose ipilimumab as first-line treatment of advanced/metastatic non-small-cell lung cancer (NSCLC). We assessed the association of efficacy with programmed death ligand 1 (PD-L1) expression and tumor mutational burden (TMB). Two hundred eighty-eight patients with previously untreated, recurrent stage IIIB/IV NSCLC received nivolumab 3 mg/kg every 2 weeks plus ipilimumab 1 mg/kg every 6 weeks. The primary end point was objective response rate (ORR) in patients with 1% or more and less than 1% tumor PD-L1 expression. Efficacy on the basis of TMB (FoundationOne CDx assay) was a secondary end point. Of treated patients with tumor available for testing, 252 patients (88%) of 288 were evaluable for PD-L1 expression and 98 patients (82%) of 120 for TMB. ORR was 30% overall and 41% and 15% in patients with 1% or greater and less than 1% tumor PD-L1 expression, respectively. ORR increased with higher TMB, plateauing at 10 or more mutations/megabase (mut/Mb). Regardless of PD-L1 expression, ORRs were higher in patients with TMB of 10 or more mut/Mb (n = 48: PD-L1, ≥ 1%, 48%; PD-L1, < 1%, 47%) versus TMB of fewer than 10 mut/Mb (n = 50: PD-L1, ≥ 1%, 18%; PD-L1, < 1%, 5%), and progression-free survival was longer in patients with TMB of 10 or more mut/Mb versus TMB of fewer than 10 mut/Mb (median, 7.1 2.6 months). Grade 3 to 4 treatment-related adverse events occurred in 29% of patients. Nivolumab plus low-dose ipilimumab was effective and tolerable as a first-line treatment of advanced/metastatic NSCLC. TMB of 10 or more mut/Mb was associated with improved response and prolonged progression-free survival in both tumor PD-L1 expression 1% or greater and less than 1% subgroups and was thus identified as a potentially relevant cutoff in the assessment of TMB as a biomarker for first-line nivolumab plus ipilimumab.