SignificanceMoxetumomab pasudotox is a fusion protein of an anti-CD22 Fv andPseudomonasexotoxin. It is highly active against leukemia in vitro but acute lymphoblastic leukemia (ALL) patients often are resistant. Studies with cultured cells showed resistance is caused by reduced diphthamide, the intracellular target ofPseudomonasexotoxin, but diphthamide is not reduced in most cells from most ALL patients. To study how resistance develops in animals, we injected ALL cells into mice and found that resistant cells occur in discrete bone marrow niches and contain major chromosomal and transcriptional changes. Mice pretreated with 5-azacytidine show greatly improved responses, supporting a trial of the combination in leukemia patients.