Aggressive cancer cells show histological similarities to embryonic stem cells. As differentiated cells can re-acquire pluripotency and self-renewal by transfection with the transcription factors OCT4, SOX2, KLF4 and MYC, with Nanog as readout for success, we comprehensively investigated their occurrence and frequency in human astrocytomas of different malignancy grades, primary and matched recurrent glioblastomas, short- and long-term glioblastoma cultures and glioma cell lines. Among astrocytomas, mRNA expression of OCT4, MYC and (less robust) KLF4 increased with malignancy, while in recurrent glioblastomas MYC expression slightly decreased. Correlation analysis revealed distinct positive correlation between distinct stem cell markers, and this effect was most prominent in the recurrent glioblastoma cohort. In situ, embryonic stem cell factors were found also in more differentiated tumor regions. Respective cells were rarely actively proliferating and showed single or combined expression signatures, which, at least in parts, corresponded to observed positive correlations of mRNA expression. However, a 'master-marker' defining the complete glioma stem cell subset could not be confirmed. In glioma cell lines, long- and short-term cultures, embryonic markers were detected at comparable levels. Upon exposure to temozolomide, increased expression of KLF4 (and lesser Nanog and OCT4) was observed. Experimental intrinsic overexpression of SOX2, KLF4 or OCT4 did not affect the other stem cell factors. The embryonic stem cell factors comprehensively investigated in this project can control self-renewal and pluripotency, and therefore tumorigenicity. They should be considered for the development of future diagnostic and therapeutic strategies.