Little is known about the long-term consequences of overexpression of the human telomerase reverse transcriptase (hTERT) gene in T lymphocytes. To address this issue, we transduced polyclonal as well as clonally derived populations of naive and memory CD4⁺ T cells from 2 healthy donors (aged 24 and 34 years) with retroviral vectors encoding green fluorescence protein (GFP) and hTERT (GFP-hTERT) or GFP alone. After transduction, cells were sorted on the basis of GFP expression and cultured in vitro until senescence. T cells transduced with hTERT exhibited high stable telomerase activity throughout the culture period. Relative to GFP controls, minor changes in overall gene expression were observed yet the proliferative lifespan of the hTERT-transduced populations was significantly increased and the rate of telomere loss was lower. Nevertheless, hTERT-transduced cells showed progressive telomere loss and had shorter telomeres at senescence than controls (2.3 ± 0.3 kilobase [kb] versus 3.4 ± 0.1 kb). Furthermore, a population of cells with 4N DNA consisting of binucleated cells with connected nuclei emerged in the hTERT-transduced cells prior to senescence. We conclude that overexpression of hTERT in CD4⁺ T cells provides a proliferative advantage independent of the average telomere length but does not prevent eventual genetic instability and replicative senescence.