App-V FAQ: Can App-V be used to run 16-bit applications on Windows x64?

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No, App-V is not a compatibility solution. App-V does not provide any additional layers that applications can use when executing on different versions of Windows. Although 16-bit applications can be deployed on 32-bit Windows via App-V, it cannot be used to run 16-bit applications on 64-bit Windows.

If 64-bit Windows does not support 16-bit applications, then neither will App-V. The lack of 16-bit application support in x64 Windows is documented in this Microsoft Knowledgebase Article: 64-bit versions of Windows do not support 16-bit components, 16-bit processes, or 16-bit applications:

The x64-based versions of the Microsoft Windows Server 2003 operating systems and Microsoft Windows XP Professional x64 Edition do not support 16-bit programs, 16-bit processes, or 16-bit components. However, these 64-bit versions of Windows may recognize some 16-bit installers and automatically convert the 16-bit installer to a 32-bit installer.