Myeloma cell suspensions from tumor-bearing mice were cultured in soft agar by a new method. Two types of colonies were detected in cultures of tumor-infiltrated spleens: 1) Monolayer colonies contained peroxidase-positive cells, presumably of hematopoietic origin, which had 40 normal chromosomes and were not malignant; 2) the cells which grew over one another to form spherical masses have been identified as tumor cells by chromosome analysis and by their ability to induce tumors that produced the original myeloma protein in BALB/c mice. The linear relationship demonstrated between the number of tumor colonies formed and the number of cells plated indicated that this cell culture method can be used to assay mouse myelomas for tumor colony-forming cells. CMRL 1066, a cell culture medium devised by Parker et al. (“Methods of Tissue Culture,” New York, Hoeber, 1961), contains an unidentified labile factor required by myeloma cells but not by hematopoietic cells. Human serum promoted and fetal calf serum inhibited myeloma cells, whereas both sera were equally good for hematopoietic cells. Irradiated myeloma-infiltrated spleen cells enhanced the plating efficiency of the myeloma cells. A mouse kidney tubule feeder layer was essential for the growth of both tumor and hematopoietic cells. With a cell separation technique, tumor stem cells forming colonies in culture were shown to belong to the same size class as those forming spleen colonies. In addition, tumor stem cells were separated from normal hematopoietic cells.