The expression of the hair follicle stem cell marker CD34 was analyzed in five different head and neck squamous cell carcinoma (HNSCC) cell lines with different antibodies. All HNSCC cell lines expressed CD34 on their cell surface. After cell cycle synchronization via serum starvation， we observed cyclic CD34 expression in HNSCC cells dependent on cell cycle progression via immunofluorescent staining and flow cytometric analysis. Investigation of the CD34(+) and CD34(-) HNSCC populations revealed most of the cells in S-phase and G2/M-Phase in CD34(+) cells in contrast to CD34(-) cells. Knockdown of CD34 in HNSCC cells led to diminished clonal expansion in a colony forming assay after subjecting the cells to ionizing radiation. Furthermore， knockdown of CD34 after cell cycle synchronization induced high CK1， CK4， and CK5 gene expression and downregulation of CK10 gene expression as shown by Taqman quantitative PCR analysis. The expression levels of CK1 and CK10 were verified via western blot analysis. In summary， our study shows that CD34 plays a role during cell cycle progression of head and neck squamous cell carcinoma and additionally is involved in irradiation resistance and differentiation of malignant oral keratinocytes.