While therapies targeting CD19 by antibodies, CAR-T cells and T cell engagers have improved the response rates in B-cell malignancies; the emergence of resistant cell populations with low CD19 expression can lead to relapsed disease. We developed an in vitro model of adaptive resistance facilitated by chronic exposure of leukemia cells to a CD19-immunotoxin. Single-cell (sc) RNAseq showed increase in transcriptionally distinct CD19^low populations in resistant cells. Mass cytometry demonstrated that CD22 was also decreased in these CD19^low resistant cells. ATAC-seq showed decreased chromatin accessibility at promoters of both CD19 and CD22 during development of resistance. Combined loss of both CD19 and CD22 antigens was validated in samples from pediatric and young adult patients with ALL that relapsed after CD19 CAR-T targeted therapy. Functionally, resistant cells were characterized by slower growth and lower basal levels of MEK activation. CD19^low resistant cells exhibited preserved B cell receptor signaling and were more sensitive to both BTK and MEK inhibition. These data demonstrate that resistance to CD19 immunotherapies can result in decreased expression of both CD19 and CD22 and can result in dependency on BTK pathways.