Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, they integrate genes in semi-random patterns, and their transduction efficiency is highly variable. In this study, several non-viral gene delivery vehicles, promoters, and additional variables were compared to optimize non-viral transgene delivery and expression in both Jurkat and primary T cells. Overall, transfecting Jurkat cells in X-VIVOTM 15 media with Lipofectamine LTX provided a high transfection efficiency (63.0±10.9% EGFP+). However, the same method yielded a much lower transfection efficiency in primary T cells (8.1±0.8% EGFP+). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans (HSPGs) detected via mRNA-sequencing. PYHIN DNA sensors (e.g., AIM2, IFI16) were also expressed at high levels in Primary T cells, which can induce apoptosis when bound to cytoplasmic DNA. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake and/or DNA sensing in the cytoplasm, so both of these factors should be considered in the development of future viral and non-viral T cell gene delivery methods.