TuneUp Disk Space Explorer reports 252,794.88 MB (excludes system and hidden files, very close to Explorer). chkdsk's total (259,641.4 MB) roughly matches Explorer's total (259,068.5 MB), TreeSize is the odd man out. chkdsk reports 64.0 MB used by the NTFS log. chkdsk reports 1055.66 MB used by the system (e.g. chkdsk reports 354.8 MB used for indexes (TreeSize will not show this). chkdsk reports 258,166.9 MB used for files. Explorer (select all files => properties) reports 254,749.0 MB used (does actual calculation, leaves out hidden files, still shows ~35GB more than TreeSize). Explorer (drive properties) reports 259,068.5 MB used (reports cached free space value, shows ~40GB more than TreeSize). TreeSize reports 219,231.0 MB allocated (1GB wasted). 
On my system (1TB drive with 4K clusters): There is a good general article about free space discrepancies in NTFS, although that primarily refers to discrepancies in what Explorer reports, and does not cover the difference between Explorer and TreeSize.
"allocated", by the way, but this would not explain your difference (nor would other answers here regarding difference in allocated space) as TreeSize reports the allocated space (meaning that TreeSize would show a higher total than the sum of the file sizes, which is what you want).
You can hold the mouse over "C:\" in TreeSize, and you will see a field for "size" vs.
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