You need to save the MXD when you're done.import arcpy, os
mxd = arcpy.mapping.MapDocument("M:/nih/AsbestosAbatement/Floorplans/BLDG_6A/Floorplanpdf.mxd")
for lyr in arcpy.mapping.ListLayers(mxd, "BuildingFloorLines"):
layer.definitionQuery = "[FLR] = '02'"
mxd.save()
print 2
If you're currently in arcmap and want to act on the active map:import arcpy, os
mxd = arcpy.mapping.MapDocument("current")
for lyr in arcpy.mapping.ListLayers(mxd, "BuildingFloorLines"):
layer.definitionQuery = "[FLR] = '02'"
arcpy.RefreshActiveView()
arcpy.RefreshTOC()
print 2