Although the ability of endogenous CD4−CD8− double negative (DN) T cells to suppress B cells has been documented, the extent to which ex vivo converted DN T cells suppress B cells activity is still being explored. The aim of this study was to determine whether and what extent ex vivo converted CD4−CD8− DN T cells suppress B cell activation and antibody production. We found that ex vivo converted DN T cells suppressed proliferation of activated B cells in a perforin and cell–cell contact dependent manner. In addition, ex vivo converted DN T cells significantly inhibited the production of IgG by stimulated B cells. This study provides evidence that ex vivo converted CD4−CD8− double negative T cells can down-regulate immune responses by suppressing B cell proliferation and IgG production, and supports efforts to develop ex vivo DN T cell therapies.