Today I ran into an issue with a recently deployed Citrix farm, which has been in use for the last couple of weeks.
I’m receiving the error “This network connection does not exist” when launching a single application. However when I change the published application to another server (B) it works fine. This message appears for all users this morning. We have 2 XenApp 5.0 FP3 servers in our farm, however when I (Domain Admin) tried to launch it on server A it worked fine. I tested again with one of the users and still had the same message.
I then duplicated the application instance and called it a new name and didn’t change any other settings apart from the server group and added server A. The user could then launch the application from server A under the new published instance. In fact the user could launch from both Server A and Server B.

I tested this a few more times with the user, both published applications point to the same location, have the same users, etc..

So it appears the problem is with the application named instance and could be a DB issue, but how can I be sure?

Now my users are happy again, I will create a test user and try to replication the problem.
A good tool for this would be Process Monitor by sysinternals, (Now Microsoft). This shows all the processes, files, and registry entires for a single user. I will be looking for permission issues or invaild entries.
More information on this subject can be found on the Citrix forum: http://forums.citrix.com/thread.jspa?threadID=258424&tstart=0