Nestin‐Cre mice are widely used to generate gene deletions in the developing brain. Surprisingly, fewNestin‐Cre lines have been characterized for their temporal and brain region‐specific recombination. In addition, some Nestin‐Cre lines express Cre outside the central nervous system, making it difficult to choose appropriate lines for targeting genes with brain region‐restricted expression. Here we describe the properties of a Nestin‐Cre transgenic line and its use for conditional deletions of Pitx2, a paired‐like homeodomain transcription factor. We report that Nestin‐Cre conditional Pitx2 mutant mice have ocular and craniofacial defects consistent with the role of human PITX2 in Rieger syndrome. Conditional mutants exhibit defects in midbrain neuronal development similar to those in Pitx2 homozygous null embryos, but lack the abnormalities in subthalamic nucleus neurons that occur with complete loss of Pitx2 function. These data indicate that normal differentiation of midbrain neurons depends upon adequate Pitx2 function during the period of active neurogenesis. genesis 44:336–344, 2006. Published 2006 Wiley‐Liss, Inc.