Establishing requirements for the survival of human immature B cells in vitro has proved elusive. In this article, we report prolonged survival of B-lineage leukemic cells on 'feeder layers' of bone marrow (BM)-derived stromal cells in a serum-free environment. In 15 of 18 cases of B-lineage acute lymphoblastic leukemia (ALL), there was a greater than 50% decrease in the number of viable cells after 72 hours of culture in medium alone. Cell loss was preceded by molecular and cellular changes characteristic of programmed cell death, or apoptosis, and was not suppressed by adding interleukin-7 to the tissue culture medium. By contrast, the use of allogeneic BM stromal cells as feeder layers prevented apoptosis in 10 of 12 cases of ALL, leading to extended survival of the blast cells. This method was not successful when the allogeneic marrow cells were replaced with established cell lines. In six of eight cases in which the numbers of intact CD19+ lymphoblasts were counted by flow cytometry after 7 days of culture, the proportion of such cells recovered in the presence of BM stromal cells was 68.8% to 124.7% (median, 95.3%) of that originally seeded, as opposed to the 0.3% to 15.9% fraction (median, 0.7%) obtained in the absence of stromal cells. Survival requirements of the B-lineage lymphoblasts appeared to be heterogeneous, as cells from 3 of the 18 cases studied showed no signs of apoptosis in serum-free tissue culture medium that lacked BM stromal cells. The only cells not giving rise to viable cultures were from two hyperdiploid (greater than 50 chromosomes) cases with identical karyotypes. The serum-free assay described here can be used to compare the survival requirements of normal and leukemic B-cell progenitors as well as to identify the molecules involved in the interaction between BM stroma and immature B cells.