Significance: Commercial strategies to deliver biomolecular cargo ex vivo (e.g., electroporation, lipofection) to clinically relevant cell lines are limited by toxicity, cost, and throughput. These technical limitations have inhibited development of these technologies into streamlined clinical platforms for manufacturing gene-modified stem cells and cancer immunotherapies. Here, we demonstrate an acoustofluidic platform capable of delivering plasmids with high throughput to human T lymphocytes, peripheral blood mononuclear cells, and CD34 ⁺ hematopoietic stem and progenitor cells. Acoustofluidic-treated cells showed evidence of cytosolic DNA delivery, endocytic DNA aggregation, and nuclear membrane rupture. Collectively, these observations demonstrate the utility of this method as a research tool for gene editing applications and mechanistic studies of plasma membrane and nuclear membrane repair.