Chromosome breakage analysis following exposure of cultured cells to DNAcrosslinking agents has long been considered the 'gold standard' for the confirmation or exclusion of Fanconi anemia (FA). Cells containing DNA damage are arrested and accumulate, with a 4c DNA content, near the S/G2-phase border of the cell cycle. As manifestation of their impaired DNA damage response, FA cells typically express elevated G2-phase cell fractions which can easily be measured by flow cytometry. In our experience with more than 3,000 such analyses, at most 1 in 10 blood samples submitted for the exclusion or confirmation of FA yields a positive result. Compared to traditional chromosome breakage analysis, cell cycle testing is less demanding and offers the advantage of speed and low cost. We prefer flow cytometric cell cycle testing for the initial screening of patients, in whom unexplained thrombocytopenia, macrocytic aplastic anemia or other clinical findings require the exclusion of FA. In addition to its application in diagnostic screening, we here show that cell cycle analysis has become a valuable tool for the determination of clastogen sensitivity (such as required within the context of complementation studies), for the precise definition of cell cycle checkpoints, and for the quantitative determination of compartment-specific cell cycle delay or cell cycle arrest. Cell cycle analysis not only provides a reliable test system for the initial confirmation or exclusion of FA, but also serves as a highly informative tool for the comprehensive characterization of the FA cellular phenotype.