Blinatumomab, a bispecific antibody that directs CD3+ T cells to CD19+ tumor cells shows variable efficacy in B-progenitor acute lymphoblastic leukemia (B-ALL). To determine tumor-intrinsic and extrinsic determinants of response, we studied 44 adults with relapsed or refractory B-ALL (including two MRD positive) treated with blinatumomab using bulk tumor and single-cell sequencing. The overall response rate in patients with hematological disease was 55%, with a high response rate CRLF2-rearranged Ph-like ALL (12/16, 75%). Pretreatment samples of responders exhibited a tumor-intrinsic transcriptomic signature of heightened immune response. Multiple mechanisms resulted in loss of CD19 expression, including CD19 mutations, CD19 mutant allele-specific expression, low CD19 RNA expression and mutations in CD19 signaling complex member CD81. Low hypodiploid patients were prone to CD19 negative relapse due to aneuploidy-mediated loss of the non-mutated CD19 allele. Increased expression of a CD19 isoform with intra-exonic splicing of exon 2, CD19 ex2part, at baseline or during therapy was associated with treatment failure. These analyses demonstrate both tumor intrinsic and extrinsic factors influence blinatumomab response. We show that CD19 mutations are commonly detected in CD19-negative relapse during blinatumomab treatment. Identification of the CD19 ex2part splice variant represents a new biomarker predictive of failure to blinatumomab therapy.