Previous studies of T cell reconstitution following allogeneic stem cell transplantation have described long-lasting T cell defects, including decreased levels of autocrine proliferating CD4+ T cells. However, T cell functions during the early phase of conditioning-induced, pre-engraftment pancytopenia have not been characterized previously. We used a whole blood assay to investigate T cell proliferation and cytokine release during the period of pre-engraftment cytopenia. The study included 13 acute leukemia patients receiving myeloablative conditioning followed by transplantation of G-CSF-mobilised peripheral blood stem cells derived from HLA-matched family donors. Maximal proliferation and cytokine release could not be reached by anti-CD3 stimulation alone, but was dependent on the presence of additional costimulation with anti-CD28. Circulating T cells showed a broad cytokine release profile after activation, and the highest levels were detected for IFNgamma, GM-CSF and IL-6. Correlation analyses showed that TNFalpha/IL-4/IL-5/IL-13 in particular were released as a separate cluster, IFNgamma and GM-CSF correlated strongly, whereas IL-17 showed a weak correlation to IL-6 only. The capacity of circulating T cells derived during pre-engraftment cytopenia to release high levels of IFNgamma, IL-6 and IL-17 in response to in vitro activation with anti-CD3+anti-CD28 showed statistically significant correlations with later acute GVHD. We conclude that allotransplanted patients have a functional T cell system even during the pre-engraftment period of severe pancytopenia.