Charge transport in organic thin film transistors takes place in the first few molecular layers in contact with the gate dielectric. Here we demonstrate that the charge transport pathways in these devices are extremely sensitive to the orientational defects of the first monolayers, which arise from specific growth conditions. Although these defects partially heal during the growth, they cause depletion of charge carriers in the first monolayer, and drive the current to flow in the monolayers above the first one. Moreover, the residual defects induce lower crystalline order and charge mobility. These results, which are not intuitively explained by electrostatics arguments, have been obtained by combining in situ real time structural and electrical characterization together with ex situ AFM measurements, on thin films of a relevant n-type organic semiconductor, N,N'-bis(n-octyl)-dicyanoperylene-3,4:9,10-bis dicarboximide grown by sublimation in a quasi-layer-by-layer mode at different substrate temperatures.