Outcomes for pediatric relapsed or refractory acute lymphoblastic leukemia (ALL) remain unsatisfactory. Administration of Inotuzumab ozogamicin (InO), a CD22-targeted antibody-drug conjugate, has shown remarkable activity but predictors of response to InO with improved accuracy over CD22 expression and site density have not been reported. We analyzed 68 samples from 28 patients enrolled in Children's Oncology Group trial AALL1621 (NCT02981628) collected before and after treatment with InO. We utilized a B-ALL-centric CyTOF protein profiling approach. Highdimensional clustering analysis depicted distinct tumor profiles between complete and partial responders. Our analyses identified frequencies of CD22high cells and CD22low/Bcl-2high cells as predictors of good and poor response, respectively. Furthermore, residual blasts at end of cycle 1 or 2 showed persistently high expression of Bcl-2 family members. These data provide new insights into predictors of InO treatment and raise the potential of Bcl-2 family inhibitors as concurrent therapy in these patients.