Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm(-/-) and p53(-/-) consistently develop thymic lymphoma early in life (1,2), and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols (1,3). Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain (1,4). We have successfully established several T-cell lines using this protocol and inbred strains ofAtm(-/-) [FVB/N-Atm(tm1Led)/J] (2) and p53(-/-) [129/S6-Trp53(tm1Tyj)/J] (5) mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.