3 and half year old really pushing ons!! any advice?

my son is 3 and a half and is actually a really nice kids lots of people comment on it he plays well with others, i run a dayhome and normally have 6 kids 3 and under hes fab with his baby sister! But just lately he is really pushing ons trying to make me mad when i tell him to not do somehting he gives me a look like f you im gonna do it! He just being really bad and i dont know how to deal with it! time out isnt working i have started to put him up to his bedroom and not let him play with everyone while hes acting up but i dont like doing this i feel its not fair and also like im telling him off all day which is stressful and so draining! its beginging to affect my marraige and is actually made us not want anymore children! which we were planning another right away! any advice will be welcomed thanks