Abstract： T cell anergy has been successfully induced under different conditions in cloned CD4 + T cells, but induction of T cell anergy in vivo has been difficult and controversial. Due to the low frequency of naturally occurring T cell population with specificity to a defined antigen, it is very difficult to study anergy of naive T cells without prior in vivo priming which complicates the interpretation of experimental data. To solve this problem, we adopted the HNT-TCR transgenic mice which have homogenous antigen specific CD4 + T cell population. In this study, we generated an influenza virus hemagglutinin (HA) peptide-specific CD4 + T cell clone from the HNT-TCR transgenic mice and induced anergy using APCs which were treated with the crosslinker, ECDI ( 1-ethyl-3-3 (3-dimethylaminopropyl) carbodiimide). The proliferative response of the cloned or freshly purified naive CD4 + transgenic T cells after treatment with ECDI-treated APCs and the HA peptide antigen was monitored as the index of anergy induction. The results showed that anergy was successfully induced in the cloned HNT-TCR transgenic CD4 + T cells. It was determined that the induced anergy was antigen- and MHC-specific. By contrast, anergy was not observed in freshly purified naive CD4 + transgenic T cells under the same conditions. The results suggest that naive CD4 + T cells may have different anergy inducing requirements, or that cloned CD4 + T cells may have certain priming or in vitro cloning artifact which makes them more susceptible to anergy induction. We propose that induction of T cell anergy may depend on the T cell growth, activation and differentiation state or cloning conditions. The results from the present study may have important implications for the study of the mechanism(s) underlying T cell anergy induction in vivo and for applications of immune tolerance based therapy.