The β1D integrin is a recently characterized isoform of the β1 subunit that is specifically expressed in heart and skeletal muscle. In this study we have assessed the function of the β1D integrin splice variant in mice by generating, for the first time, Cre-mediated exon-specific knockout and knockin strains for this splice variant. We show that removal of the exon for β1D leads to a mildly disturbed heart phenotype, whereas replacement of β1A by β1D results in embryonic lethality with a plethora of developmental defects, in part caused by the abnormal migration of neuroepithelial cells. Our data demonstrate that the splice variants A and D are not functionally equivalent. We propose that β1D is less efficient than β1A in mediating the signaling that regulates cell motility and responses of the cells to mechanical stress.