The Australian Multi-centre study on CD34+ cell quantitation by flow cytometry documented, first, the extent of variation of CD34+ cell enumeration and, second, the influence of flow cytometric gating on CD34+ cell measurement. A PBSC harvest analyzed by 20 participating centers showed results ranging from 0.64% to 2.80%, with a median of 1.54% CD34+ cells. Of 20 centers, 9 obtained results within +/-10% of the median (the criteria for reproducibility suggested by the ISHAGE Guidelines). The flow cytometric gating strategy was identified as one of the major variables among the methods used. In stage 2, list mode data from two samples were analyzed by 24 Australian and overseas centers, including the authors of three published guidelines. Significantly different CD34+ results were obtained when different gating strategies were used (p < 0.006). When all the centers used the same gating strategy, the measurement of CD34+ cells fell within a narrow range, with 0-7% of results outside +/-10% of the median. However, when different gating strategies were used, the results were more widely scattered, with 17% of centers outside +/-10% of the median. This study demonstrated the potential impact of flow cytometric gating strategy on the reproducibility of CD34+ cell enumeration.