Rare tumor cells with stem cell characteristics, termed cancer stem cells (CSC) or tumor-initiating cells (TIC), have been found to play a major role in tumor formation and metastasis. In this work I used the spheroid culture method to enrich and further characterize such cells from a parental clear cell renal cell carcinoma (ccRCC) cell line. Two slightly different methods for spheroid generation, resulting in two morphologically different spheroid subtypes, were evaluated with respect to their stem cell properties. The stability as well as the plasticity of spheroid-derived cell lines compared to adherently grown cells was investigated. For this purpose, spheroids were re-cultured long-term under conditions of adherent growth and assayed for retention of investigated characteristics. Growth characteristics, such as long-term proliferative potential, growth in „soft agar assay“ and spheroid-forming efficiency in the „neurosphere assay“ over long-term passaging were assayed to assess and confirm stem cell features in vitro. Also functional assays (ALDH activity, „side population assay“) were evaluated for suitability to enrich CSC from the investigated cell lines. Besides, the ability to generate differentiated progeny was tested by using in vitro differentiation assays for adipogenic and osteogenic lineages. Additionally, the expression of known stem cell markers and EMT markers was examined by flow cytometric immunophenotyping, and, to gain a more comprehensive view, also by mRNA Sequencing. While the results of those experiments strongly support the CSC character of spheroids as well as the presence of low amounts of CSC already in the parental cell line, no tumor formation in vivo was observed. The results obtained in this work do not fit a simple straightforward model, and it will take complex algorithms to reach deeper into the understanding of the complex phenomena involved. Nonetheless, the heterogeneity and plasticity observed in the investigated cell lines fits the current view on heterogeneity and plasticity of CSC. Due to their reflection of different aspects of CSC, such as expression of surface markers, EMT status, differentiation potential, and growth characteristics, the cell lines obtained and characterized in this work may serve as one more tool to unravel the basic mechanisms of RCC tumor formation.