Pitx2 is a homeodomain transcription factor that is mutated in Rieger syndrome, a haploinsufficiency disorder affecting eyes and teeth. Pitx2 also has a postulated role in left-right axis determination. We assessed the requirements for Pitx2 directly by generating hypomorphic and null alleles. Heterozygotes for either allele have eye abnormalities consistent with Rieger syndrome. The ventral body wall fails to close in embryos homozygous for the null allele, leaving the heart and abdominal organs externalized and the body axis contorted. In homozygotes for either allele, the heart tube undergoes normal, rightward looping and the stomach is positioned normally. In contrast, homozygotes for both alleles exhibit right isomerization of the lungs. Thus, Pitx2 is required for left-right asymmetry of the lungs but not other organs. Homozygotes for either allele exhibit septal and valve defects, and null homozygotes have a single atrium proving that a threshold level of Pitx2 is required for normal heart development. Null homozygotes exhibit arrest of pituitary gland development at the committed Rathke pouch stage and eye defects including optic nerve coloboma and absence of ocular muscles. This allelic series establishes that Pitx2 is required for the development of mulitple organs in a dosage-sensitive manner.