Impairment of adipogenesis contributes to the development of obesity-related insulin resistance. The current in vitro approaches for its assessment represent crude estimates of the adipogenic potential because of the disruption of the in vivo microenvironment. A novel assessment of in vivo adipogenesis using the incorporation of the stable isotope deuterium ((2)H) into the DNA of isolated adipocytes and stroma-vascular fraction from adipose tissue has been developed. In the current study, we have refined this technique by purifying the adipocytes via a negative immune selection and sorting the plastic adherent stroma-vascular (aSV) subfraction (using 3 h culture) that contains mostly adipocyte progenitor cells and ∼10% of small adipocytes. Using a 3-week 8% (2)H(2)O ingestion with a high-fat diet (HFD) or HFD plus pioglitazone (HFD-P), we demonstrate that the fractions of new aSV cells (f(aSV)) and immunopurified adipocytes (f(AD)) (the ratio of their (2)H-enrichment of DNA to the maximal (2)H-enrichment of DNA of bone marrow reference cells) recapitulate the known hyperplastic mechanism of weight gain with pioglitazone treatment. We conclude that f(aSV) and f(AD) are reliable indices of in vivo adipogenesis. The proposed method represents a valuable tool for studying the effect of interventions (drugs, diets, and exercise) on in vivo adipogenesis.