We have produced two lines of transgenic mice in which the expression of temperature-sensitive SV-40 large T antigen is targeted to bone marrow megakaryocytes via the platelet factor 4 (PF4) tissue-specific promoter. The progeny of these transgenic mice were observed for about 3 mo, and no malignancies were detected over this period of time. The offspring of these transgenic mice, 6- to 12-wk of age, served as a source of bone marrow cells, which upon in vitro cultivation at the permissive temperature yielded immortalized cell lines (MegT). At the permissive temperature, MegT cells exhibit the characteristics of early 2N and 4N megakaryocytes which include the presence of specific gene products such as PF4, glycoprotein IIb, acetylcholinesterase, and CD45 as well as the absence of molecular markers of other cell lineages such as the macrophage marker Mac-1, the T helper cell marker CD4, the mast cell marker IgE, the T cell marker CD2 or the erythroid cell marker alpha-globin. The inactivation of the oncogene by a shift of temperature from 34 degrees to 39.5 degrees C produces a reduction in the frequency of the 2N cells, in conjunction with the appearance of 8N and 16N cells, consisting of 27 and 3% of total cells, respectively. Thus, we have generated hematopoietic cell lines that are trapped in the early stages of megakaryocyte commitment, but able to undergo part of the normal program of terminal differentiation.