To whom was Hashem referring when He told Moshe that the men who were after his blood had died?
Rashi and Targum Yonasan: He was referring to Dasan and Aviram, who had lost all their money, and a poor man 1 is considered like dead.
Rashbam and Seforno: Pharaoh and his servants who had tried to execute him and from whom he had fled. 2
Having already been instructed to go to Egypt and having received permission from Yisro to leave, why did Hashem see fit to issue him with fresh instructions to leave?
Ramban: Initially, he thought that his life was in danger, and he therefore planned to go on his own clandestinely. Hashem now informed him that his life was safe and that he had nothing to fear, and that he should therefore return to Egypt to stay with the people until the exodus. 1
When exactly, did this episode take place?
Rashi writes that Dasan and Aviram had become poor, so they are called dead. Perhaps they became Metzora'im, blind, or childless. Also they are called dead!
Da'as Zekenim (1), Hadar Zekenim (1), Moshav Zekenim, Rosh: All blemishes were cured at Matan Torah, but returned after the Egel. In Korach's rebellion, we find that they were in Machaneh Yisrael, unlike Metzora'im. "Will you gouge out these people's eyes?" connotes that they were not blind, and they had children 1 - "u'Vneihem v'Tapam."
Da'as Zekenim (2), Hadar Zekenim (2): Hashem told Moshe why he need not fear. If they were wealthy, even if they were called dead for another reason, they could hire someone to kill Moshe, (or give bribes and influence the king - Rosh)!
Da'as Zekenim, Hadar Zekenim: If they were destined to beget children later, they would not be called dead due to being childless now. (Further, if Korach's rebellion was shortly after the Meraglim (or before, like the Bartenura and Mizrachi infer from Rashi Devarim 1:1), they could not beget infants and [older] children within two years. However, perhaps they were Metzora'im, and later healed? - PF)