We developed a mouse model of acute T/natural killer (NK) acute lymphoblastic leukemia (ALL) that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We demonstrated the non-random nature of methylation in the transformation from benign polyclonal expansion to leukemia, and identified a novel tumor suppressor gene silenced in the majority of human leukemia but not solid tumors. To this, we utilized restriction landmark genomic scanning (RLGS) on eight IL-15 transgenic (tg) mice with T/NK ALL, and performed RLGS on an additional five spleens from IL-15tg mice without ALL, but with polyclonal T/NK expansion, and on four spleens from wild type (wt) mice. A total of 2447 RLGS fragments with good resolution on each RLGS profile were analyzed. Only 1–2 variable changes were detected in either wt or polyclonal T/NK expansion controls. In contrast, the eight spleens with clonal T/NK leukemia had 45 to 209 changes (1.8 to 8.5% of total fragments) consistent with aberrant DNA methylation. The association of RLGS fragment losses or reduced intensity with leukemic transformation versus polyclonal expansion was highly significant (P<0.001). The promoter of Inhibitor of DNA binding 4 (Id4) was found methylated in 87.5% of the mouse T/NK ALLs. ID4 was found methylated in 85.7% (72/84) of primary human acute myeloid leukemias (AML) and in 100% (61/61) of chronic lymphocytic leukemias (CLL). Id4 was silenced secondary to promoter methylation in 100% mouse T/NK ALLs (n=5) and human acute leukemias (n=6). Overexpression of Id4 induced apoptosis in mouse cell line YAC-1 and inhibited tumor growth both in vitro and in vivo. These data, and an analysis of 219 RLGS profiles from different types of human tumors demonstrate that the ID4 promoter is preferentially methylated and functions as a putative novel tumor suppressor gene in mouse and human leukemia. This study highlights the use of a novel animal model, novel mouse NotI-EcoRV arrayed library, and RLGS to examine the role of methylation in leukemic transformation and to identify new targets for prevention and therapeutic strategies.