Rob, write.FCS was trying to replace empty value for each keyword in order to conform to FCS3.0. The replacement command sub("^$"," ",y) apparently mess up the original spillover matrix. I just fixed it by skipping the replacement for spillover keyword. The fix (version 1.23.7) should appear in BioC development branch in one day. Optionally you can check out from https://github.com/RGLab/flowCore. Thanks. -Mike