There are many known adipokines differentially secreted from the different adipose depots; however, their paracrine and autocrine effects on de novo adipocyte formation are not fully understood. By developing a coculture method of preadipocytes with primary subcutaneous and visceral adipocytes or tissue explants, we could show that the total secretome inhibited preadipocyte differentiation. Using a proteomics approach with fractionated secretome samples, we were able to identify a spectrum of factors that either positively or negatively affected adipocyte formation. Among the secreted factors, Slc27a1, Vim, Cp, and Ecm1 promoted adipocyte differentiation, whereas Got2, Cpq, interleukin-1 receptor-like 1/ST2-IL-33, Sparc, and Lgals3bp decreased adipocyte differentiation. In human subcutaneous adipocytes of lean subjects, obese subjects, and obese subjects with type 2 diabetes, Vim and Slc27a1 expression was negatively correlated with adipocyte size and BMI and positively correlated with insulin sensitivity, while Sparc and Got2 showed the opposite trend. Furthermore, we demonstrate that Slc27a1 was increased upon weight loss in morbidly obese patients, while Sparc expression was reduced. Taken together, our findings identify adipokines that regulate adipocyte differentiation through positive or negative paracrine and autocrine feedback loop mechanisms, which could potentially affect whole-body energy metabolism.