The responsiveness of developing NK cells is set depending on the MHC environment in which they mature. (a) Experimental scheme for producing mixed fetal liver cell chimeras and testing rejection of MHC I–deficient cells. WT (+), β2m^−/− (−), or a 1:1 mix of WT and β2m^−/− (mix) fetal liver cells was used to reconstitute lethally irradiated and NK-depleted hosts. (b) After 9–17 wk of reconstitution, chimeras were reirradiated or not before testing whether the chimeras rejected β2m^−/− spleen cells or bone marrow cells, as indicated. Unmanipulated B6 and β2m^−/− mice were tested in parallel as controls for rejection and tolerance, respectively. The rejection assay was performed by injecting CFSE^high-labeled β2m^−/− cells (spleen or bone marrow) mixed with an equal number of CFSE^low-labeled WT spleen cells as an internal control. Rejection of β2m^−/− spleen cells was determined by flow cytometry of spleen cells 18 h later. Some groups of chimeras were pretreated i.p. twice (on days −2 and −1) with 200 µg PK136 (NK1.1) antibody (NK depleted), as indicated. Data represent means ± SEM (n = 2–5 mice). The experiment was performed three times with spleen cell targets and once with bone marrow cell targets. nd, not done.