Sublethal damage to red blood cells (RBCs) during extracorporeal life support (ECLS) may lead to RBC loss. Using flow cytometry, phosphatidylserine-positive (PhS+) RBCs and RBC extracellular vesicles were quantified as measures of sublethal RBC injury in 41 pediatric ECLS runs, stored RBC units, and normal adult subjects. We estimated the clearance half-life of PhS+ RBCs and compared the rates of RBC loss during pediatric ECLS due to phlebotomy, intravascular hemolysis, and extravascular clearance of PhS+ RBCs. Extracorporeal life support patients had 0.9% PhS+ RBCs, sixfold higher than normal subjects (p < 0.0001). Phosphatidylserine-positive RBCs were increased in stored RBC units (twofold in whole blood derived units, p = 0.0013; 12-fold in apheresis RBC units, p < 0.0001). Phosphatidylserine-positive RBCs were cleared with an average half-life of 15 hours. During ECLS, PhS+ RBC clearance accounted for 7% of RBC loss (1–60%), phlebotomy 12%, and intravascular hemolysis 12%. Increasing PhS+ RBCs occurred in 40% of patients that died on ECLS. Red blood cell extracellular vesicles, another marker of red cell injury/activation, were elevated fivefold during ECLS. Phosphatidylserine exposure on RBCs is increased during ECLS, marking these cells for extravascular clearance with a half-life of ~15 hours and accounting for ~7% of RBC loss.