Dear Gautam,

your problem suggests that your cell line is not able to form extracellular matrix efficiently in later stage of differentiation or when the cell number increases. It means some of the proteins like metalloproteins are deficit when they are required in large number. The proteins necessary for the ECM formation directly or indirectly is utilized in early stage of differentiation but lack after certain point of growth.

I guess heat inactivation of the serum may not be exact problem. you might have to add some extra supplement that support the ECM formation. Since heat inactivation is required to avoid immunoglobulin aggregation, so it might be necessary step but not mendatory. However, you can check to see what happens when you do not heat-inactivate your serum, how your cells respond to it. Or you can start with the heat-inactivated serum and add small amount of non-heat-inactivated serum in the later stage.

However, the answer to your question might be found out at:

http://bitesizebio.com/23706/heat-inactivation-of-serum-for-tissue-culture-is-it-necessary/

Mature adipocytes spend a lot of energy on the maintenance of the ECM. ECM remodeling is mediated by a balanced constructive and destructive enzymes together with their enhancers and inhibitors. ECM remodeling is an energy costing process regulated by insulin, by the energy metabolism. Overgrowth of adipocytes may lead to instability of the ECM, possibly mediated by hypoxia. SO it is also possible that the cells lack energy for the construction of ECM. Since cells in early stage of differentiation have already used the energy what you had provided.

find out more:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2839497/

Thanks Anshu for your answer and the links which you have given. I will go through it  and let you know in case of any doubt. 

no, it is not necessary,   you can add rosiglitazone to your induction media. it work great  with me 

Article Protocol for effective differentiation of 3T3-L1 cells to adipocytes

Article Glucagon-like peptide 1 (GLP-1)-based therapy upregulates LX...

I agree that it is not necessary.  I my master degree thesis we did not used. Please see Fig 1 in:

http://www.sciencedirect.com/science/article/pii/S0014299908003531

 Thank you Yonghan and Fausto for your helpful answers.

We regularly batch test FBS as not all serum induce good differentiation. This we found is a key to a good 3T3L1 adipocyte differentiation!