T.D.
Okay...I have a few thoughts. I don't think it is a delayed reaction to the new room.
First, I think you might be on to something with the whole not playing in his room thing. Try that for a little while.
Second, be very careful on how you are responding to him and your emotions when you are tucking him in. I know that it is difficult to be calm when you are worried about him, but you need to be VERY self-assured when you are putting him to bed. Kids "learn" to fear based off of our reactions to things. If you act like there is something wrong, then he will think there is something to be afraid of. For example, my kids are not scared of the dark. However, we read a book talking about various emotions and one of them is "scared" and it shows a picture of a little boy afraid of a dark thunderstorm. That night, my daughter tried to pretend to be scared of the dark. I know that she was pretending because earlier in the day she pretended to be angry, be sad, be silly and all the other emotions. So I calmly told her that was just a book and we weren't going to pretend to be scared. She stopped immediately. Had I reacted differently, I am sure she would have become scared of the dark. We purposefully skip that page when reading that book now and she has never said she was scared of the dark again. So basically what I am saying is, watch your emotions. Make sure you are very calm and straightforward and cheerful "Its time for bed now. I love you. I'll see you in the morning. Good night."
Third, make sure he is not "overtired". When my kids don't get a good enough nap, they have a harder time falling asleep.
Finally, I had a similar situation happen with my 2 year old girl when she was around that age. The first time it happened, I did overreact. She was probably just overtired, but I was worried she was in pain or something. So I sat in there with her for 3 hours. She liked that. She played that game with me for a little over a week. Finally I decided enough was enough. I calmly told her it was bed time and I would see her in the morning. I gave her motrin just to make sure she wasn't in pain from teething. Then I left. I let her cry for 20 minutes. Then I sent my husband in. She is not so excited about having Daddy in there--I guess he's not as comforting or as fun or something. Anyway, he went in and CALMLY told her that she needed to be quiet, "it is night night time. Go to sleep." He kissed her and then left. She cried for another 20 minutes or so and went to sleep. The next night she cried for maybe 10 minutes. The next night I didn't give her any motrin and she cried for maybe 5 minutes.
I would like to point out, that I am NOT a cry it out fan. Up until that point I had not let her cry it out for more than a few minutes when I knew she was exhausted. I have 3 other children and I have never let any of them cry it out. However, we had gotten into a little pattern that she liked. She knew I would come in there if she screamed really hard so that's what she did.
So, with all that said, my best advice for you is trust your instincts. If you are pretty sure there is nothing physically wrong with him, and he seems fine the rest of the time, he has probably just developed a habit that you need to help him break--however you choose to do that. He is your son...you know what will work best for him. Good luck!