We identified apilimod as an antiproliferative compound by high-throughput screening of clinical-stage drugs. Apilimod exhibits exquisite specificity for phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) lipid kinase and has selective cytotoxic activity in B-cell non-Hodgkin lymphoma (B-NHL) compared with normal cells. Apilimod displays nanomolar activity in vitro, and in vivo studies demonstrate single-agent efficacy as well as synergy with approved B-NHL drugs. Using biochemical and knockdown approaches, and discovery of a kinase domain mutation conferring resistance, we demonstrate that apilimod-mediated cytotoxicity is driven by PIKfyve inhibition. Furthermore, a critical role for lysosome dysfunction as a major factor contributing to apilimod’s cytotoxicity is supported by a genome-wide CRISPR screen. In the screen, TFEB (master transcriptional regulator of lysosomal biogenesis) and endosomal/lysosomal genes CLCN7, OSTM1, and SNX10 were identified as important determinants of apilimod sensitivity. These findings thus suggest that disruption of lysosomal homeostasis with apilimod represents a novel approach to treat B-NHL.