• CD4+CD25+Foxp3+ donor T cells, including those that were alloantigen-specific, preferentially reconstituted after PTCy.• CD4+CD25+Foxp3+ donor T cells played a necessary role in GVHD prevention by PTCy, but did not appear sufficient to prevent severe and fatal GVHD.• Foxp3+ donor T cells were necessary for GVHD prevention by PTCy both at early and late post-transplant time points, but appeared increasingly important as time progressed.• The suppressive mechanisms induced early after PTCy were sufficient to prevent new donor T cells from causing GVHD. This evidence concerns the gene CD4 and graft versus host disease.