Obesity is one of the major causes of metabolic diseases such as diabetes and heart attack, and, hence, can lead to low quality of life. Elaborating adipocyte differentiation is very crucial for formulating the treatment and prevention of obesity. The objective of this study is to investigate the seeding cell number required to obtain optimum lipid accumulation during adipocyte differentiation using the 3T3-L1 cell line. Two sets of 5.48×104 (for Day 0 and Day 8 of differentiation), of 10.96×104 (for Day 8) and of 21.92×104 (for Day 8) of 3T3-L1 cells were seeded in each wells of a 12-well plate. Isobuthylmethylxanthine (IBMX), Dexamethasone, and Insulin-containing differentiation cocktails was added into the medium at Day 0 for 48 hours. The medium was changed every two days. Day 0 and Day 8 samples were then stained using Oil Red O and were examined under the microscope to observe the lipid droplets (red-coloured). The lipid droplets were quantified by measuring the absorbance at wavelength of 550 nm. In the study, seeding the number of 10.96×104 cells produced very significantly higher lipid accumulation, as compared with seeding the number of 5.48×104 cells. However, doubling the seeding number into 21.92×104 cells did not increase the lipid droplets significantly. This study found that the optimum seeding number to obtain the maximum lipid droplets during 3T3-L1 adipocyte differentiation was 10.96×104 cells.