Several cytogenetic lesions in chromosomes 2, 5, 12, and 16 have been repeatedly coselected with in vitro macrophage differentiation in a clonal murine thymic tumor cell line. Parental-type subclones, which show an extremely immature hemopoietic phenotype, do not carry the aberrations. The frequency of the stable differentiated variants is elevated by 5-azacytidine and bromodeoxyuridine, consistent with chromosome breakage being responsible for the phenotype. The frequency is also raised by dexamethasone. Since variants are 300-3,000-fold more resistant to dexamethasone than parental clones, we interpret this to be largely due to selection. Three of the lesions, on chromosome 2, match those previously described as associated specifically with in vivo-generated murine myeloid tumors, induced by X irradiation and corticosteroid treatment. Several implications follow from these observations. (1) In vitro differentiation in clonal tumor cell lines can be used to select for tumor-associated lesions. This should allow genetic and molecular analysis of the chromosome 2 lesions and of others that may pinpoint genes critical to macrophage differentiation and transformation. (2) Myeloid and lymphoid tumors that occur in response to X irradiation may diverge from a common initiating tumor. (3) The hemopoietic lineage switch phenomenon, previously described by several authors, may be caused by similar or identical chromosome aberrations.