Leukemia and lymphoma cells are potential targets in cancer therapy for genetic manipulation either by transgene expression or silencing of endogenous gene expression. In addition, genetically engineered autologous lymphocytes expressing a chimeric antigen against a receptor overexpressed in tumor or tumor vasculature are promising cellbased therapeutics for cancer. The major hurdle to the successful implementation of these attractive approaches is the lack of a smart device for efficient transgene delivery and expression in the lymphocytes. Recently, we developed an efficient nanocarrier of carbonate apatite for intracellular delivery and release of DNA molecules, achieving very high level of transgene expression in primary as well as cancer cell lines. However, its efficacy in human T leukemia cells is comparatively low. Here, we reveal that simultaneous stimulation of human T leukemia cells by the most commonly used phorbol ester-based protein kinase C (PKC) activator and an actin filament disrupting agent dramatically enhanced carbonate apatite-mediated transgene delivery and expression in the cells by synergistically activating protein kinase C (PKC), while rapidly extruding Ca2+ of intracellularly dissolved particles through plasma membrance-associated Ca2+- ATPase. Moreover, endocytosis of the DNA-associated particles across the cell membrance was found to follow the clathrin-dependent route in both normal and activated cells. The findings thus offer significant insights for pre-clinical and clinical ex vivo trials of cancer treatment utilizing autologous T cells.