Glycerol 3-phosphate acyltransferase-1 (GPAT-1) catalyzes the initial and rate-limiting step in de novo glycerophospholipid and triacylglycerol (TAG) biosynthesis. We have previously shown that peripheral T cell proliferation and cytokine production is altered in GPAT-1 gene-ablated (KO) mice. This finding is important in light of the reduction in GPAT-1 activity associated with aged T cells. To determine if the mechanism for altered peripheral T cell function is linked to altered T cell development, we assessed thymic function in 3, 6 and 16-week old GPAT-1 KO compared to wild type (WT) mice. At 16 weeks of age, there was a significant reduction in thymic T cell production in KO compared to WT mice but not at 6 weeks of age. The reduced thymic T cell production was associated with altered thymic development as confirmed by increased numbers of double-negative (DN) thymocytes and a significant reduction in the double positive (DP) thymocytes suggesting a developmental block at the DN stage. This change was accompanied by an increase in the single positive CD4 subset. These changes were associated with reduced glycerophospholipid mass while thymic cortex and medulla architecture was not altered by GPAT-1 KO. Taken together, these data suggest that GPAT-1 deletion is capable of reducing the number of new T cells produced via alterations in membrane receptor function rather than by causing deleterious changes within the thymic microenvironment explaining in part the observed alterations in peripheral T cell function.