Influenza is one of the most common respiratory diseases in humans. The response to vaccination is frequently poor in immunosuppressed individuals. The aim of the present study was to develop an enzyme-linked immunospot (ELISPOT) assay for measuring of the specific T-cell response to influenza vaccination. In all, 18 healthy subjects and six stem cell transplantation (SCT) patients tested before and 4 weeks after influenza vaccination were included in the present study. Peripheral blood lymphocytes were stimulated with four influenza peptides; three based on sequences from the hemagglutinin and one from the M1 protein. The ELISPOT assay and the measurement of intracellular IFN-γ production were used to determine the cell-mediated responses after stimulation with the peptides. Influenza vaccination elicited strong cell-mediated immune responses in the healthy controls to all four peptides with 3.2–6.9-fold increases in the number of IFN-γ producing spots/10 6 cells. By intracellular staining, it was suggested that CD4+ cells mediated the responses to the hemagglutinin peptides. In contrast, there was no increase in the number of IFN-γ producing cells response after vaccination in the six SCT patients. In conclusion, our results suggest that the ELISPOT assay might be used as a complement to serology for monitoring of future influenza vaccine studies in SCT patients.