Burkholderia pseudomallei is the causative agent of melioidosis. While adaptive immunity has been shown to be important for host resistance to B. pseudomallei, the direct interaction of the bacteria with adaptive immune cells such as T and B cells is not well known. To address this question, we infected Jurkat T cells, as well as human primary CD4(+) and CD8(+) T cells, with live B. pseudomallei. We found that live bacterial infection could costimulate T cells to produce interleukin-2 (IL-2) and gamma interferon (IFN-gamma) in the presence of anti-CD3 cross-linking antibodies. Bacterial supernatant could also costimulate T cells, and this was due to the presence of flagellin in the supernatant. However, T cells infected with bacterial mutants lacking flagellin showed strong impairment in IL-2 but only a slight impairment in IFN-gamma production. When cross-linking of CD3 is replaced by IL-2, live bacterial infection was still able to costimulate human primary T cells to produce IFN-gamma and flagellin is only a minor ligand contributing to this costimulation. Thus, live B. pseudomallei could potentially costimulate T cells not only in an antigen-specific manner but also in a nonspecific manner through bystander activation via IL-2.