Functional analysis of gene candidates and testing of novel therapeutics in Wilms tumors (WT) has been hampered by the lack of in vitro model systems. WT are characterized by a spectrum of histological appearances, but published cell lines are mostly derived from rare anaplastic variants or even non-WT. There has been some success in establishing primary cultures, but these are often poorly characterized or only derived from less frequent WT1 mutant tumors. We report the generation of a set of primary WT-cell cultures using a simple cultivation protocol. Our cultures could be established after preoperative chemotherapy and irrespective of histological subtypes or genetic alterations. The presence of tumor-specific genetic alterations validates these cultures as being tumor-derived. Genetic characterization is of utmost importance as some cultures with similar morphological appearance lacked such alterations and either represent clonal variants or normal cells. By immunohistochemistry, the cells are either epithelial or more mesenchymal, and the latter exhibiting a longer life span with 30 or more passages before undergoing senescence. This may be related to WT being embryonal tumors with a strong differentiation potential that may prevail in vitro. Telomeres progressively shorten with cultivation, but their length does not predict lifespan. hTERT transfection may partly allow establishment of immortalized lines, because 2/7 cultures avoid senescence even in later passages. Importantly, these cells can be efficiently manipulated by transfection, making them a useful model system for in vitro testing. © 2011 Wiley Periodicals, Inc.