Binding of CD80/86 to CD28 is regarded as the main T cell costimulatory interaction. However, CD28 downregulates soon after T cell activation. To investigate potential cross-interaction between CD137 (4-1BB) and CD28, we stimulated T cells with anti-CD3 in the presence of A549 lung carcinoma cells expressing CD80/CD86 and 4-1BBL molecules, transduced into the cells using recombinant non-replicating adenoviruses. Following initial T cell proliferation, the proportion of CD28(+) cells in both CD4(+) and CD8(+) populations was rapidly reduced by CD80/86 costimulation, whereas cultures costimulated with just 4-1BBL continued to express CD28. CD28 was also downregulated in cultures costimulated with both CD80/86 and 4-1BBL. Interestingly, in cells costimulated with CD80/86 that had downregulated CD28 expression and ceased to proliferate, reactivation of proliferation by 4-1BBL costimulation also restored their CD28 expression. These findings show a positive effect of CD137 signalling on CD28 expression, similar to the effect of CD28 engagement on 4-1BB expression during the initial phases of T cell activation. Moreover, they point to the importance of signals through 4-1BB for the purposes of ex-vivo T cell activation and expansion.