Abstract Body: Atrial septation is integral for the separation and functions of the mammalian pulmonary and systemic circulations. Atrial septal defects (ASDs) are common, affecting around 0.2% of live births. Mutations affecting the transcription factor TBX5 are known to drive the formation of ASDs in humans and mice. However, the molecular mechanisms underpinning atrial development and septation, as well as how they may be perturbed in ASDs and the role of TBX5 in this process remain unclear. Here we analyze the cell populations that contribute to the atrial septum during murine heart development, and examine the role of Tbx5 in atrial septation. Utilizing the Tbx5^del/+ model, which leads to ASDs with a high penetrance, we performed single-cell RNA sequencing of control Tbx5^+/+and mutant Tbx5^del/+ embryonic atria across a timecourse of development. Tbx5^del/+ hearts displayed a significantly reduced proportion of atrial cardiomyocytes at embryonic day 11.5, with a relative shift to give rise to fewer Pitx2-positive and more Bmp10-positive atrial cardiomyocytes. We further identified dysregulated genes and pathways associated with reduced Tbx5 dosage, including components of several key signaling pathways such as BMP and Wnt, known to play roles in cardiac development, and members of the semphorin and thrombospondin family. Further work will aim to understand how alterations of these signaling pathways contribute to the formation of ASDs. Together these findings define Tbx5-dependent transcriptional programs regulating atrial cardiomyocyte development and provides mechanistic insight into Tbx5-associated ASDs. This work is funded by the AHA (25POST1376966), NIH (5R01HL114948, 5R01HL155906), Additional Ventures, and the Younger Family Fund.