Blinatumomab is an efficacious immunotherapeutic agent in B cell acute lymphoblastic leukemia (B-ALL). However, the pharmacogenomic basis of leukemia response to blinatumomab is unclear. Using genome-wide CRISPR, we comprehensively identified leukemia intrinsic factors of blinatumomab sensitivity, i.e., the loss of CD58 as a top driver for resistance, in addition to CD19. Screening 1639 transcription factor genes, we then identified PAX5 as the key activator of CD58. ALL with the PAX5 P80R mutation also expressed the lowest level of CD58 among 20 ALL molecular subtypes in 1988 patients. Genome editing confirmed the effects of this mutation on CD58 expression and blinatumomab sensitivity in B-ALL, with validation in patient leukemic blasts. We described a PAX5-driven enhancer at the CD58 locus, which was disrupted by PAX5 P80R, and the loss of CD58 abolished blinatumomab-induced T cell activation with global changes in transcriptomic/epigenomic program. In conclusion, we identified previously unidentified genetic mechanisms of blinatumomab resistance in B-ALL, suggesting strategies for genomics-guided treatment individualization.