Invasive aspergillosis remains a serious complication in patients undergoing allogeneic stem cell transplantation. Since it became clear that lymphocytes provide a critical secondary defense against fungi, adoptive transfer of functionally active anti-Aspergillus T cells might be an option to restore adaptive immune effector mechanisms. Using the interferon (IFN)-gamma secretion assay, we isolated human activated T cells upon stimulation with a cellular extract of Aspergillus fumigatus. After a culturing period for 14 days, we could characterize these cells as T(H)1 cells, which also proliferated upon restimulation. The generated cells responded upon stimulation with antigens of A. flavus, A. niger and Penicillium chrysogenum, but not upon activation with Alternaria alternata and Candida albicans. In addition, the cultivated T cells were able to induce damage to A. fumigatus hyphae and showed a reduced alloreactivity compared to unselected CD4+ T cells. We further established a clinical-scale generation of anti-Aspergillus T cells. However, before performing clinical trials, open questions such as which patient population will benefit from adoptive immunotherapy with anti-Aspergillus T cells have to be addressed.