The aim was to develop T cell costimulatory molecules that are broadly applicable to augment anti-tumor immune responses upon application of a virus-modified tumor vaccine to cancer patients. We generated recombinant bispecific single-chain antibodies with one specificity directed against the CD3 or the CD28 antigen on human T cells and the other against the viral target molecule hemagglutinin-neuraminidase (HN) of Newcastle Disease Virus (NDV). By re-directing unstimulated primary human T cells against HN-expressing NDV-infected tumor cells, the bispecific molecule bsHN-CD3 cross-linked effector and target cells and rapidly induced cytotoxicity at nanomolar concentrations. The bsHN-CD28 molecule exerted T cell co-stimulatory function. Maximal T cell activation was achieved with tumor cells infected by NDV and modified with both new stimulatory molecules. This was revealed by T cell proliferation, upregulation of CD69 and CD25 and by release of cytokines, interferons and chemokines. The new molecules combine high-effectivity with specificity and safety.