Cell lines are crucial to elucidate mechanisms of tumorigenesis and serve as tools for cancer treatment screenings. Therefore, careful validation of whether these models have conserved properties of in vivo tumors is highly important. Thyrocyte-derived tumors are very interesting for cancer biology studies because from one cell type, at least five histologically characterized different benign and malignant tumor types can arise. To investigate whether thyroid tumor-derived cell lines are representative in vitro models, characteristics of eight of those cell lines were investigated with microarrays, differentiation markers, and karyotyping. Our results indicate that these cell lines derived from differentiated and undifferentiated tumor types have evolved in vitro into similar phenotypes with gene expression profiles the closest to in vivo undifferentiated tumors. Accordingly, the absence of expression of most thyrocyte-specific genes, the nonresponsiveness to thyrotropin, as well as their large number of chromosomal abnormalities, suggest that these cell lines have acquired characteristics of fully dedifferentiated cells. They represent the outcome of an adaptation and evolution in vitro, which questions the reliability of these cell lines as models for differentiated tumors. However, they may represent useful models for undifferentiated cancers, and by their comparison with differentiated cells, can help to define the genes involved in the differentiation/dedifferentiation process. The use of any cell line as a model for a cancer therefore requires prior careful and thorough validation for the investigated property.