The production of multipotential hemopoietic stem cells (CFUs) in cultures of murine bone marrow cells is supported by medium conditioned by concanavalin A-stimulated T cell hybridoma 123 or spleen cells. We present physiochemical evidence that this activity of these conditioned media, which we have operationally termed CFUs-stimulating activity (CFUs-SA), is due to a new T cell-derived lymphokine. CFUs-SA was shown by gel filtration to reside in a distinct fraction corresponding to a mean apparent m.w. of 29,000, and was distinguishable from T cell growth factor, not only by its lower apparent m.w. but also by chromatography using Phenyl-Sepharose. After isoelectric focusing of neuraminidase-treated conditioned medium, CFUs-SA was found between isoelectric points 5 and 8. In this respect, CFUs-SA appeared similar to another factor, P cell-stimulating factor, which stimulates the growth of persisting (P) cells (homogeneous populations of cells resembling mast cells) and that occurred together with CFUs-SA in both the spleen and hybridoma-conditioned media. Isoelectric focusing separated CFUs-SA from granulocyte-macrophage colony-stimulating factor, which focused as a single peak with an isoelectric point around 5.0. Thus CFUs-SA is due to a T cell-derived factor that is separable from T cell growth factor and T cell-derived granulocyte-macrophage colony-stimulating factor, but has similar properties to P cell-stimulating factor.