The paucity of cell culture models for childhood brain tumors prompted us to establish pediatric cell lines for use in biological experiments and preclinical developmental therapeutic studies. Three cell lines were established, CHLA-200 (GBM), CHLA-259 (anaplastic medulloblastoma) and CHLA-266 (atypical teratoid rhabdoid tumor, AT/RT). Consistent with an AT/RT origin, CHLA-266 lacked INI1 expression and had monosomy 22. All lines had unique DNA short tandem repeat "fingerprints" matching that of the patient's tumor tissue and were adherent on tissue culture plastic, but differed in morphology and doubling times. CHLA-200 had a silent mutation in TP53. CHLA-259 and CHLA-266 had wild-type TP53. All three lines were relatively resistant to multiple drugs when compared to the DAOY medulloblastoma cell line, using the DIMSCAN fluorescence digital image microscopy cytotoxicity assay. RNA expression of MYC and MYCN were quantified using RT-PCR (Taqman). CHLA-200 expressed MYC, DAOY and CHLA-259 expressed MYCN, and CHLA-266 expressed both MYCN and MYC. CHLA-200 was only tumorigenic subcutaneously, but CHLA-259 and CHLA-266 were tumorigenic both subcutaneously and in brains of NOD/SCID mice. Immunohistochemistry of the xenografts revealed GFAP staining in CHLA-200 and PGP 9.5 staining in CHLA-259 and CHLA-266 tumors. As expected, INI1 expression was lacking in CHLA-266 (AT/RT). These three new cell lines will provide useful models for research of pediatric brain tumors.