The failure of engraftment in human cases of in utero hematopoietic cell transplantation (IUHCT) in which no immunodeficiency exists suggests the presence of an unrecognized fetal immune barrier. A similar barrier in murine IUHCT appears to be dependent on the chimerism level and is poorly explained by a lack of T-cell tolerance induction. Therefore, we studied the effect of the chimerism level on engraftment and host natural killer (NK)–cell education in a murine model of IUHCT. The dose of transplanted cells was found to exhibit a strong correlation with both the engraftment rate and chimerism level. More specifically, a threshold level of initial chimerism (> 1.8%) was identified that predicted durable engraftment for allogeneic IUHCT, whereas low initial chimerism (< 1.8%) predicted a loss of engraftment. NK cells taken from chimeras above the “chimerism threshold” displayed durable calibration of alloresponsive Ly49A receptors and tolerance to donor antigens. Depletion of recipient NK cells stabilized engraftment in low-level chimeras (< 1.8%). These studies illustrate the importance of the early chimerism threshold in predicting long-term engraftment and host NK-cell tolerance after in utero transplantation.