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I've run through the Tutorial and first level a few times and have been unable to lock onto anyone. Whether or not I click the middle mouse button, whether or not it's on "View" mode, I don't seem to be having any luck.

Is there an alternate setting, or a trick of some kind I'm missing?
This question / problem has been solved by Jakkarimage
You're not missing a trick. Really, all you do is click down the middle mousebutton, which should translate to 'mouse3' - I don't believe you can change the controls, though it's worth searching for a config or ini file in the install folder.

Do you have an unusual mouse or a nonstandard OS?
Apparently "unusual mouse" was the answer. Just had to go in and check my middle-mouse button settings, which was set to something ridiculous. Thanks!
Glad it worked =) You can play without lockon but it really helps during high speed door breaches for a satisfyingly clean job. Particularly when throwing a knife to take down a marksman after door-mashing his friend.
After the last update I have the same problem. The lock worked fine before, I actually completed the game with it. Now nothing happens when I click the scroll wheel. Anyone else experiencing this?

On a side note, locking does work on the 360 pad. But that's not really related.
I didn't even know that there was a lock on feature

been getting through the game without using it.