L3MBTL1, a paralogue of Drosophila tumor suppressor lethal(3)malignant brain tumor (l(3)mbt), binds histones in a methylation state-dependent manner and contributes to higher order chromatin structure and transcriptional repression. It is the founding member of a family of MBT domain-containing proteins that has three members in Drosophila and nine in mice and humans. Knockdown experiments in cell lines suggested that L3MBTL1 has non-redundant roles in the suppression of oncogene expression. We generated a mutant mouse strain that lacks exons 13-20 of L3mbtl1. Markedly reduced levels of a mutant mRNA with an out-of-frame fusion of exons 12 and 21 were expressed, but a mutant protein was undetectable by Western blot analysis. L3MBTL1(-/-) mice developed and reproduced normally. The highest expression of L3MBTL1 was detected in the brain, but its disruption did not affect brain development, spontaneous movement, and motor coordination. Despite previous implications of L3mbtl1 in the biology of hematopoietic transcriptional regulators, lack of L3MBTL1 did not result in deficiencies in lymphopoiesis or hematopoiesis. In contrast with its demonstrated biochemical activities, embryonic stem (ES) cells lacking L3MBTL1 displayed no abnormalities in H4 lysine 20 (H4K20) mono-, di-, or trimethylation; had normal global chromatin density as assessed by micrococcal nuclease digests; and expressed normal levels of c-myc. Embryonic fibroblasts lacking L3MBTL1 displayed unaltered cell cycle arrest and down-regulation of cyclin E expression after irradiation. In cohorts of mice followed for more than 2 years, lack of L3MBTL1 did not alter normal lifespan or survival with or without sublethal irradiation.