The most primitive engrafting hematopoietic stem cell has been assumed to have a fixed phenotype, with changes in engraftment and renewal potential occurring in a stepwise irreversible fashion linked with differentiation. Recent work shows that in vitro cytokine stimulation of murine marrow cells induces cell cycle transit of primitive stem cells, taking 40 h for progression from G₀ to mitosis and 12 h for subsequent doublings. At 48 h of culture, progenitors are expanded, but stem cell engraftment is markedly diminished. We have investigated whether this effect on engraftment was an irreversible step or a reversible plastic feature correlated with cell cycle progression. Long-term engraftment (2 and 6 mo) of male BALB/c marrow cells exposed in vitro to interleukin (IL)-3, IL-6, IL-11, and steel factor was assessed at 2–4-h intervals of culture over 24–48 h using irradiated female hosts; the engraftment phenotype showed marked fluctuations over 2–4-h intervals, with engraftment nadirs occurring in late S and early G₂. These data show that early stem cell regulation is cell cycle based, and have critical implications for strategies for stem cell expansion and engraftment or gene therapy, since position in cell cycle will determine whether effective engraftment occurs in either setting.