I have a 2wd 2001 T4R auto and have been experiencing a vibration above 55 mph under acceleration. Once I let off the gas it mostly goes away. Fluids are good and not sure what to look into next.

Check the u joints

+ 1 on the u joints.

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Not just u-joints. There is a good chance the whole rear driveshaft needs replacement. The 2WD prop shaft has a rubber vibration dampener/coupler that wears out and requires a complete shaft replacement to fix.

It *could* just be u-joints, but be prepared for it to be the whole shaft.

-Charlie

X3 ujoints. You also state 2wd and your avatar appears lifted...if so depending on how much lift you've done your slip yoke may not fully seated on the output shaft causing it to vibrate and wobble at higher speeds when the drive shaft is rotating faster. If u joints are good then measure drive line angles make sure they match. Then disconnect your drive shaft and slide it all the way in till it bottoms out and measure the space between the diff and drive shaft...if you got an inch or more of space you may need a drive shaft spacer. For what it's worth if you have an inch of space when the slip yoke is bottomed out don't get a 1 inch spacer...you dont want your yoke bottomed out. If it's an inch gap maybe 3/4 spacer so your skip yoke still has some movement. You're just trying to get the slip seated well again.

You can some times get vibration just from road conditions. To rule this out what I did was jack the rear wheels up with the 4runner pointed in a safe direction place jack stands under the rear wheels and chock the front wheels. Slowly throttle the engine up to the offending speed allowing the transmission to shift. As you try different things for me this was a time saver. I had a rebuilt drive shaft that had u-joints that were the wrong size if you replace them only use Spicer or Toyota I don't have the part number at hand. What I found out was the cross was to small allowing the drive line to move when up and down not in the normal failure of bad caps. Even after replacing the Drive line it was still out of balance so I was able to balance it using a hose clamp method (very old school but I am up to 20,000 mile things are still great)

Thanks for all the info.
I had some time to look at it and 1 of the U joint caps were destroyed. Im not sure if the entire shift is bad as it did not look cracked. This is my mom's car and I asked how long it has been doing this her answer was not sure but I can hear it over my radio so its annoying now. Hahaha....
The T4r in my pic is my 2000 4wd.
Thanks again for the info.

In January I installed a driveshaft from this firm

Toyota Replacement Driveshafts

into a 2WD Gen3 4Runner, automatic transmission, 4-cylinder engine (can't recall its year).

The results were good; my friend's son was pleased. If this drive shaft's bearing wear out, they're standard Spicer bearings which are widely available.