Abstract Nonstimulated fetal liver (FL) from 14.5-day gestation mice had no natural killer (NK) cell activity and <3% expressed NK1.1. Even after short-term (3–4 day) culture of FL with the late-acting cytokines, interleukin (IL)-15 or IL-2, little or no NK activity was detected. However, longer-term (13 day) culture with IL-2 plus stroma derived from bone marrow (BM) of adult mice, resulted in extensive proliferation and differentiation to mature NK cells. Cell numbers began to increase after 4 days, and by day 13, they had increased 40-fold and 69% of the cells were NK1.1 + with high NK activity and 5%–10% were NK1.1 -B220 + . With stroma, but no IL-2, equivalent proliferation occurred, but differentiated cells were predominantly NK1.1 -B220 + , not NK cells. Culture for 13 days without stroma, but with either IL-2, IL-15, FLTK3-ligand (L) or stroma-conditioned medium, resulted in less than fivefold expansion, and minimal NK activity. Culture with combinations of FLTK3-L or ckit-L plus IL-15 or IL-2 increased both cell number and NK activity, but the increase in cell number was less than that seen with stroma plus IL-2. By limiting dilution assay on stroma plus IL-2, the precursor frequency was 1/(2660 ± 292) whole FL cells and the absolute number, but not the frequency, increased during culture on stroma without IL-2. The NK cell progenitors were found in sorted NK1.1 − and Sca-1 + c-kit + lineage - subpopulations at a frequency of 1/(156 ± 52.5). Together, these data suggest that the NK lineage cells in FL are primarily in early stages of development. They are highly proliferative, respond to early acting cytokines and express stem cell markers.