•Truncating mutations in MS4A1 with subsequent antigen loss is a major mechanism of resistance to CD3 × CD20 bispecific antibodies.•Spatial heterogeneity and branching evolution underlie progression in lymphoma during CD19 and CD20 targeting immunotherapy. Display omitted CD19 chimeric antigen receptor (CAR) T cells and CD20 targeting T-cell–engaging bispecific antibodies (bispecs) have been approved in B-cell non-Hodgkin lymphoma lately, heralding a new clinical setting in which patients are treated with both approaches, sequentially. The aim of our study was to investigate the selective pressure of CD19- and CD20-directed therapy on the clonal architecture in lymphoma. Using a broad analytical pipeline on 28 longitudinally collected specimen from 7 patients, we identified truncating mutations in the gene encoding CD20 conferring antigen loss in 80% of patients relapsing from CD20 bispecs. Pronounced T-cell exhaustion was identified in cases with progressive disease and retained CD20 expression. We also confirmed CD19 loss after CAR T-cell therapy and reported the case of sequential CD19 and CD20 loss. We observed branching evolution with re-emergence of CD20+ subclones at later time points and spatial heterogeneity for CD20 expression in response to targeted therapy. Our results highlight immunotherapy as not only an evolutionary bottleneck selecting for antigen loss variants but also complex evolutionary pathways underlying disease progression from these novel therapies. Duell et al traced aggressive and indolent B-cell lymphomas as the tumors escaped elimination by T-cell–redirecting immunotherapy in patients. The authors showed that truncating mutations in MS4A1 with subsequent CD20 antigen loss are a major mechanism of resistance to CD3×CD20 bispecific antibodies. However, there was considerable spatial and temporal heterogeneity in target antigen expression during relapses, including reemergence of CD20 expressing clones. These data indicate complex branching evolutionary trajectories under the selection pressure of serial immunotherapy.