Background: Biallelic loss of function variants in AGPAT2, encoding 1-acylglycerol-3-phosphate O-acyltransferase 2, cause congenital generalized lipodystrophy type 1, a disease characterized by near total loss of white adipose tissue and metabolic complications. Agpat2 deficient (Agpat2−/−) mice completely lacks both white and interscapular brown adipose tissue (iBAT). The objective of the present study was to characterize the effects of AGPAT2 deficiency in brown adipocyte differentiation. Methods: Preadipocytes obtained from newborn (P0.5) Agpat2−/− and wild type mice iBAT were differentiated into brown adipocytes, compared by RNA microarray, RT-qPCR, High-Content Screening (HCS), western blotting and electron microscopy. Results: 1) Differentiated Agpat2−/− brown adipocytes have fewer lipid-laden cells and lower abundance of Pparγ, Pparα, C/ebpα and Pgc1α, both at the mRNA and protein levels, compared those to wild type cells. Prmd16 levels were equivalent in both, Agpat2−/− and wild type, while Ucp1 was only induced in wild type cells, 2) These differences were not due to lower abundance of preadipocytes, 3) Differentiated Agpat2−/− brown adipocytes are enriched in the mRNA abundance of genes participating in interferon (IFN) type I response, whereas genes involved in mitochondrial homeostasis were decreased, 4) Mitochondria in differentiated Agpat2−/− brown adipocytes had altered morphology and lower mass and contacting sites with lipid droplets concomitant with lower levels of Mitofusin 2 and Perlipin 5. Conclusion: AGPAT2 is necessary for normal brown adipose differentiation. Its absence results in a lower proportion of lipid-laden cells, increased expression of interferon-stimulated genes (ISGs) and alterations in mitochondrial morphology, mass and fewer mitochondria to lipid droplets contacting sites in differentiated brown adipocytes.