Despite clear evidence that a local accumulation of auxin is likewise critical for floral organ initiation than for vegetative tissues, much less is known about the molecular key players that regulate auxin-controlled flower development. Here, by an analysis of physiological and morphological parameters and by a spatial and temporal dissection of auxin fluxes and expression of key players of ABCB-mediated auxin transport in the Arabidopsis flower, we demonstrate a crucial role for the FKBP42, TWISTED DWARF1 (TWD1), in the regulation of flower development. Our analyses revealed that TWD1 promotes flower shape and number, stamen elongation, pollen maturation, nectary functionality and seed development. Most of the described developmental defects in twd1 are shared with the abcb1 abcb19 mutant, which can be attributed to the fact that TWD1 as a described ABCB chaperon is a positive regulator of ABCB1 and ABCB19-mediated auxin transport. We predict an overall housekeeping function for ABCB1 during earlier stages, while ABCB19 seems to be responsible for the key event of rapid elongation at later stages of stamen development. Our data indicate that TWD1 controls flower development by differential activation of ABCB-mediated auxin transport.