Abstract Body: Perivascular adipose tissue (PVAT) regulates vascular function, and alterations in its vasoactive role are part of the pathogenesis of cardiovascular diseases. Thoracic PVAT possesses a dynamic adipocyte population that changes with development, aging, and disease states. Currently, the ontogeny and developmental time points of PVAT adipocytes remain poorly understood. Lineage tracing indicates thoracic PVAT adipocytes derived from vascular smooth muscle cells Sm22a and myogenic progenitors Myf5 as early as embryo day 8.5. Generally, it is known that the vascular system forms before brown adipose tissue; however, there is still no consensus on when PVAT adipocytes appear during development and how this is compared to non-PVAT sites. We hypothesize that PVAT adipocytes begin to appear after embryonic day 8.5. Male and female adipochaser mice (Adipoq-rtTA-TRE-Cre-tdTomato n=5) were used to trace adipocytes (tdT+). Pregnant female mice were fed a doxycycline-containing chow diet during embryonic days E7-E14 (Stage I) or E14-E18 (Stage II) and kept on a regular chow diet thereafter. Sections from thoracic (TA) PVAT and interscapular brown AT (iBAT) were immunolabeled with anti-RFP: AF568, anti-PLIN1:AF488, and DAPI. Image acquisition was done in a confocal Eclipse Ti2-E AXR microscope and analysis in Qupath v0.4.4. Mature adipocytes positive to tdT were quantified as nuclei tdT+/PLIN1+ (reported as % of total nuclei). A lower percentage of adipocytes were developed in the TA (15.4%±2.2) compared to iBAT (80.4%±4.3) in stage I. Similarly, in stage II TA adipocytes (17.06%±5.07) were lower than iBAT (79.9%±8.04) in stage II. No differences were found between males (15.3%±3.2) and females (15.5%±3.2) of stage I or males (12.57%±4.1) and females (26.02%±2.01) of stage II. These findings indicate that PVAT develops as early as gestational day 7 at a slower rate than non-PVAT depot. This supports the notion of distinct adipocyte developmental pathways between PVAT and non-PVAT depots, offering new insights into how these differences may influence responses to aging and cardiovascular disease. Future studies will study adipocyte development during the early or later stages of pregnancy and how these changes are in different regions of PVAT.