Despite considerable advances in understanding the molecular pathogenesis of Wilms' tumor (WT), its cell biology is less well understood, partly due to the paucity of established WT cell lines. We report here the establishment of a new anaplastic WT cell line, 17.94, which expressed NCAM, SALL1, and CITED1-phenotypic features expected of metanephric blastema-derived cells. Treatment of 17.94 cells with 12-O-Tetradecanoylphorbol 13-acetate caused morphological changes, which led to reduced NCAM and SALL1 expression, but expression of vimentin was maintained, indicating a potential for stromal differentiation. The 17.94 cell line contained a TP53 mutation, consistent with the anaplastic histology of the original tumor, but lacked mutations in WT1, WTX, or CTNNB1, which are the other genes involved in WT pathogenesis. The 17.94 cells showed no loss of heterozygosity at 7p, 11p, or 16q; however, DNA hypermethylation was detected at several loci, including the H19 differentially methylated region (indicative of loss of imprinting of IGF2 at 11p15) and at the PCDH@ gene clusters at 5q31. The derivation of the 17.94 cell line should help to further dissect the genetic-epigenetic interactions involved in the pathogenesis of WT.