Immune dysregulation contributes to the pathogenesis of pediatric acute liver failure (PALF). Our aim is to identify immune activation markers (IAMs) in PALF that are associated with a distinct clinical phenotype and outcome. Among 47 PALF study participants, 12 IAMs collected ≤ 6 days after enrollment were measured by flow cytometry and IMMULITE assay on blood NK and CD8+ lymphocytes and subjected to unsupervised hierarchical analyses. A derivation cohort utilizing 4 of 12 IAMs which were available in all participants (% perforin(+) and % granzyme(+) CD8 cells, absolute number of CD8 cells, soluble interleukin-2 receptor level) were sufficient to define High (n=10), Medium (n=15), and Low IAM (n=22) cohorts. High IAM was more frequent among those with indeterminate etiology than those with defined diagnoses (80% vs 20%, p<0.001). High IAM associated with higher peak serum total bilirubin levels than low IAM (median peak TB of 21.7 vs 4.8 mg/dL; p<0.001) and peak coma grades. 21-day outcomes differed between groups with liver transplantation more frequent in high IAM participants (62.5%) than those with medium (28.2%) or low IAM (4.8%) (p=0.002); no deaths were reported. In an independent validation cohort (n=71) enrolled in a prior study, segregation of IAM groups with etiology, initial biochemistries, and short-term outcomes were similar, although not statistically significant. High serum aminotransferases, total bilirubin levels and leukopenia at study entry predicted a high immune activation profile. Conclusion: Four circulating T-lymphocyte activation markers identify a subgroup of PALF participants with evidence of immune activation associated with a distinct clinical phenotype and liver transplantation. These biomarkers may identify PALF participants eligible for future clinical trials of early targeted immunosuppression.