John Calvin Commentary Exodus 4:23

"and I have said unto thee, Let my son go, that he may serve me; and thou hast refused to let him go: behold, I will slay thy son, thy first-born." — Exodus 4:23 (ASV)

And I say to you, Let my son go. This was not the beginning of the mission, but its final clause; for Moses warned the desperate man of his son’s death when everything else had been tried in vain.

The meaning is, then, that the obstinacy of the tyrant must not prevent Moses from pressing him even to this final act. Therefore, this injunction was an exhortation to perseverance, as appears from the context when God declares that he will punish the obstinacy of the tyrant because he refused to obey the command to let the people go. Moreover, since this denunciation was very severe and might greatly awaken the tyrant’s wrath, Moses was therefore commanded so early to prepare himself lest he fail in this matter.