It was a gradual process, removing DOS.

- Windows 3.1 and earlier ran on DOS and relied on DOS for all its file I/O (although graphics, keyboard etc, were implemented by Windows drivers not BIOS or DOS ones)
- Windows 3.11 for workgroups added a 32-bit kernel and added the *option* to do direct I/O from Windows ("32 bit file access") - although this option was not on by default and sometimes caused problems
- Windows 95 turned "32 bit file access" on by default and it worked a lot better - they added the ability to multitask windows apps (as well as DOS ones, which wfw311 could do)
- Windows 98 essentially changed nothing
- Windows ME is a bit weird, I don't know a lot about it

At the same time

Windows NT 3.1 was developed from OS/2 essentially, with IBM. It used no DOS at all, and only implemented a "DOS box" - where you could run some DOS commands.
Windows NT 4 I think added a better DOS box where you could run more DOS things, but it was still only an emulation.
Windows 2000 and Windows XP were built on the NT kernel, hence did not use DOS.

The old DOS defragmenters will only be able to unfragment DOS filesystems, f.e. FAT12 and FAT16, *not* FAT32 or NTFS