Introduction. It has been previously shown that immunoregulatory DX5(+)NKT cells are able to prevent colitis induced by CD4(+)CD62L(high) T lymphocytes in a SCID mouse model. The aim of this study was to further investigate the underlying mechanism in vitro. Methods. CD4(+)CD62L(high) and DX5(+)NKT cells from the spleen of Balb/c mice were isolated first by MACS, followed by FACS sorting and cocultured for up to 96 h. After polyclonal stimulation with anti-CD3, anti-CD28, and IL-2, proliferation of CD4(+)CD62L(high) cells was assessed using a CFSE assay and activity of proapoptotic caspase-3 was determined by intracellular staining and flow cytometry. Extrinsic apoptotic pathway was blocked using an unconjugated antibody against FasL, and activation of caspase-3 was measured. Results. As previously shown in vivo, DX5(+)NKT cells inhibit proliferation of CD4(+)CD62L(high) cells in vitro after 96 h coculture compared to a CD4(+)CD62L(high) monoculture (proliferation index: 1.39 +/- 0.07 vs. 1.76 +/- 0.12; P = 0 0079). The antiproliferative effect of DX5(+)NKT cells was likely due to an induction of apoptosis in CD4(+)CD62L(high) cells as evidenced by increased activation of the proapoptotic caspase-3 after 48 h (38 +/- 3% vs. 28 +/- 3%; P = 0 0451). Furthermore, DX5(+)NKT cells after polyclonal stimulation showed an upregulation of FasL on their cell surface (15 +/- 2% vs. 2 +/- 1%; P = 0 0286). Finally, FasL was blocked on DX5(+)NKT cells, and therefore, the extrinsic apoptotic pathway abrogated the activation of caspase-3 in CD4(+)CD62L(high) cells. Conclusion. Collectively, these data confirmed that DX5(+)NKT cells inhibit proliferation of colitis-inducing CD4(+)CD62L(high) cells by induction of apoptosis. Furthermore, DX5(+)NKT cells likely mediate their cytotoxic and proapoptotic potentials via FasL, confirming recent reports about iNKT cells. Further studies will be necessary to evaluate the therapeutical potential of these immunoregulatory cells in patients with colitis.