Abstract Inhibitors of Bruton tyrosine kinase (BTK) and phosphatidylinositol 3-kinase δ (PI3Kδ) that target the B-cell receptor (BCR) signaling pathway have revolutionized the treatment of chronic lymphocytic leukemia (CLL). Mutations associated with resistance to BTK inhibitors have been identified, but limited data are available on mechanisms of resistance to PI3Kδ inhibitors. Here we present findings from longitudinal whole-exome sequencing of cells from patients with multiply relapsed CLL (N = 28) enrolled in trials of PI3K inhibitors. The nonresponder subgroup was characterized by baseline activating mutations in MAP2K1, BRAF, and KRAS genes in 60% of patients. PI3Kδ inhibition failed to inhibit ERK phosphorylation (pERK) in nonresponder CLL cells with and without mutations, whereas treatment with a MEK inhibitor rescued ERK inhibition. Overexpression of MAP2K1 mutants in vitro led to increased basal and inducible pERK and resistance to idelalisib. These data demonstrate that MAPK/ERK activation plays a key role in resistance to PI3Kδ inhibitors in CLL and provide a rationale for therapy with a combination of PI3Kδ and ERK inhibitors.