I need to run a 16-bit diagnostic tool in real mode under Windows XP, Vista, or 7. How can I accomplish this?
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Windows XP and later (Vista, 7) are based on the Windows NT kernel and don't rely on DOS, unlike Windows 3.1, 3.11, 95, 98, and ME. Windows Me dropped real-mode DOS support.
Windows NT includes a NTVDM (NT Virtual DOS Machine) that emulates a 16-bit DOS mode for 32-bit but not 64-bit versions.
If you really need to run tools that rely upon low level BIOS and hardware interface calls in 16-bit mode, you need to boot into a DOS environment completely outside of Windows.
FreeDOS an example of a separate 16-bit real mode DOS environment. You can install it into a separate partition, run it off of a live CD, or from a USB stick.